The hosts fell to a narrow defeat in their group opener against Roma, while the French champions swept past Galatasaray on matchday one.
Match preview
Wolfsburg were forced to go through Women's Champions League qualifying after they lost out to Bayern Munich in last season's Frauen-Bundesliga title race.
Their second-round qualifying tie against Fiorentina proved to be a formality, as Tommy Stroot's side produced a free-scoring display to ease into the main draw with a 12-0 aggregate victory.
Unfortunately for Wolfsburg, they were unable to repeat that sort of dominance in last week's group opener against Italian champions Roma.
Manuela Giugliano's first-half penalty proved enough to condemn Die Wolfinnen to a narrow 1-0 victory, leaving them with work to do in the next five matchdays if they are to reach the quarter-finals for the 12 time in 13 seasons.
Wolfsburg will head into Thursday's fixture in a positive mood after Vivien Endemann and Lineth Beerensteyn found the net to fire their side to a crucial 2-0 win over title rivals Bayern Munich.
After ending Bayern's 44-game unbeaten league run, Stroot's charges will now look to record a fifth consecutive victory in all competitions.
Lyon are hoping to make another bid to add to their eight European titles after they fell short in last season's final against Barcelona.
Former Juventus Women's boss Joe Montemurro has been tasked with leading Lyon to European glory after replacing Sonia Bompastor in the summer.
Montemurro has made a promising start to his tenure, having seen his side win all five of their matches in all competitions, including a successful outing in last week's Women's Champions League clash with Galatasaray.
Kadidiatou Diani netted either side of a Vanessa Gilles effort to fire Lyon to a 3-0 home victory, ensuring they have scored a minimum of three goals in each of their five matches this season.
They will now turn their focus to Thursday's tricky away trip, where they will look to extend their unbeaten away run in European competition to 11 matches.
They can also take confidence from the fact they have won each of their last three meetings with Wolfsburg in regulation time, including a 3-1 victory in their most recent encounter in the 2019-20 Women's Champions League final.
Wolfsburg Women Women's Champions League form:
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W
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Wolfsburg Women form (all competitions):
Lyon Women Women's Champions League form:
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Lyon Women form (all competitions):
Team News
Wolfsburg are without the injured quarter of Caitlin Dijkstra, Diana Nemeth, Camilla Kuver and Kristin Demann.
Endemann could make her second European start of the season after finding the net in Saturday's crucial league win.
Beerensteyn came off the bench to score the second goal against Bayern, and the Netherlands international is likely to be recalled for the midweek away trip.
As for the visitors, Selma Bacha remains unavailable for selection after sitting out the last six matches through injury.
After making some changes at the weekend, Montemurro could revert to the side that started the 3-0 win over Galatasaray.
Diani will continue to offer one of Lyon's main goal threats, having scored five goals in her last five competitive appearances.
Wolfsburg Women possible starting lineup:
Frohms; Wilms, Hendrich, Hegering, Linder; Minge, Lattwein; Endemann, Huth, Brand, Beerensteyn; Popp
Lyon Women possible starting lineup:
Endler; Carpenter, Gilles, Renard, Svava; Marozsan, Van de Donk, Horan; Diani, Chawinga, Dumornay