The hosts claimed a 2-1 victory over Switzerland in their inaugural battle in the top tier, while La Roja were forced to accept a 1-1 draw with old foes Portugal.
Match preview
Masking the disappointment of another failed World Cup Qualifying campaign by earning promotion to the highest division of the Nations League, the Czech Republic were indebted to a massive stroke of luck in their opening battle versus Switzerland.
After Noah Okafor had cancelled out Jan Kuchta's effort on the stroke of half time, a cross from Jakub Jankto only found the back of Djibril Sow, whose name then appeared next to the dreaded own goal tag after his 25-yard deflection beat Yann Sommer.
Jaroslav Silhavy's side may count themselves fortunate to be at the top of the rankings after the opening matchday, but their bid to remain in League A for a second successive cycle has got off to the best possible start for the Euro 2020 quarter-finalists.
Speaking of fast starts, Thursday's victory saw the world's 33rd-ranked nation open the scoring in a home Nations League match for the sixth time on the bounce, as well as representing their third straight success in all tournaments at their Prague headquarters.
Not since September 2020 have Silhavy's men suffered defeat of any kind on their own turf, and they are yet to draw a game in the Nations League since its inception, but the home crowd would surely accept a point versus La Roja.
Seemingly fending off interest from Manchester United with ease, Luis Enrique's preparations for the 2022 World Cup did not get off to the brightest start in Seville, as Iberian neighbours Portugal left it late to rescue a point in a 1-1 stalemate.
With Cristiano Ronaldo watching on from the bench, a defensive error from Portugal allowed Pablo Sarabia to find Alvaro Morata for the opening goal after 25 minutes, but substitute Ricardo Horta bagged his first-ever strike for his country eight minutes from time to ensure a share of the spoils.
Spain fell at the final hurdle to France in the 2020-21 Nations League, but Enrique was left satisfied with his side's endeavours in Seville on Thursday evening - the fifth draw in a row between Portugal and La Roja - and one which leaves Enrique's men third in the embryonic standings.
Sunday's game marks the first of a double-header with the Czech Republic for Spain before the month is up, and Enrique's side have now found the back of the net in each of their last 15 games in all tournaments, although they are yet to leave an away game in the Nations League with a clean sheet to their name.
However, La Roja seem to possess the correct defensive formula against the Czech Republic, who have failed to score in four of their five meetings with the 2010 world champions and are still waiting for their first win in this fixture as an independent nation.
Czech Republic Nations League form:
Czech Republic form (all competitions):
Spain Nations League form:
Spain form (all competitions):
Team News
The continued absence of Bayer Leverkusen talisman Patrik Schick is doing the Czech Republic no favours, but Silhavy at least has no fresh concerns to report from the victory over Switzerland.
The hosting manager would ideally like to keep changes to a minimum despite the 72-hour turnaround, as Adam Hlozek - who recently joined Schick at Leverkusen from Sparta Prague - features in a wide area once more.
Ondrej Lingr and Ales Mateju are both waiting in the wings should fresh legs be required, and West Ham United lynchpin Tomas Soucek prepares to earn his 50th national cap at senior level.
Meanwhile, Spain boss Enrique lost Aymeric Laporte and Thiago Alcantara to injury before the draw with Portugal - as well as coping without Eric Garcia - but La Roja came through the first matchday unscathed.
Gavi drew plenty of plaudits for his performance and ought to be fine to go again here, but it would not be surprising to see Koke and Rodri drafted into the engine room amid the packed schedule.
Ansu Fati will also hope to earn some minutes on Sunday after acting as an unused substitute in midweek, but Sarabia will expect to hold his place on the left from the opening whistle.
Czech Republic possible starting lineup:
Vaclik; Krejci, Zima, Brabec; Coufal, Soucek, Sadilek, Zeleny; Jankto, Kuchta, Hlozek
Spain possible starting lineup:
Simon; Carvajal, D. Llorente, P. Torres, Alba; Gavi, Rodri, Koke; F. Torres, Morata, Sarabia
We say: Czech Republic 0-2 Spain
The absence of several star names did not impact the Czech Republic's chances of a positive result on matchday one, but it almost certainly will in a fixture that they have traditionally struggled to get the upper hand in.