While the hosts are seeking to maintain their top-flight status for next season, the visitors are hoping to return to Germany's top tier for the first time in five years.
Stuttgart's three-year stay in the Bundesliga remains at risk of coming to an end despite picking up five points in their final three games of the regular season.
A 1-1 home draw with Bayer Leverkusen was followed by a thumping 4-1 away victory over Mainz 05 in their penultimate fixture of the campaign, and Sebastian Hoeness's side knew that another victory in their final match against relegation rivals Hoffenheim would guarantee their survival.
However, Stuttgart were forced to settle for a 1-1 draw on home soil last weekend, with Ihlas Bebou's 75th-minute opener cancelled out just five minutes later by substitute Tiago Tomas, and they ended the campaign occupying 16th place – the relegation playoff position – finishing just one point adrift of safety.
Hoeness arrived at Stuttgart in early April with the club sitting rock bottom of the Bundesliga, but he has given Die Roten a fighting chance of survival after leading them to three wins and four draws in nine matches.
History favours the Bundesliga side in the relegation playoffs, as the top-tier club have retained their status in 18 of the last 24 seasons, which bodes well for Stuttgart ahead of Thursday's first leg.
Hamburg narrowly missed out on automatic promotion from 2.Bundesliga and have been consigned to the playoffs for a second successive year despite winning 1-0 away against Sandhausen last weekend.
On a dramatic final day in the second tier, Hamburger supporters were on the pitch celebrating at full time after learning that Heidenheim were trailing 2-1 heading into stoppage time in their final match against Jahn Regensburg.
However, Heidenheim remarkably scored twice in added time, including a 99th-minute winner, to come from behind and snatch all three points, jumping from third to first to clinch the title and automatic promotion with virtually the final kick of their season.
Tim Walter is now tasked with attempting to lift his understandably deflated Hamburger side as they seek to avoid a repeat of last season's playoff defeat to Hertha Berlin, losing 3-0 on aggregate.
Hamburger finished the regular 2.Bundesliga season with the best away record, accumulating 30 points from their 17 matches on the road, although they head into Thursday's first leg having lost nine of the last 13 visits to Stuttgart, including a 3-2 defeat in May 2020 in which they let a two-goal first-half lead slip.
Borna Sosa recovered from illness to start and play the full 90 minutes last weekend, and the creative Croatia international, who has provided seven assists so far this season, is set to continue at left wing-back, with Josha Vagnoman set to operate on the opposite flank.
However, the visitors are able to welcome back Bakery Jatta and Ludovit Reis from one-match suspensions, and Reis could return to the first XI at the expense of either Sonny Kittel or Jean-Luc Dompe, the latter of whom scored the only goal in last weekend's win over Sandhausen, though.
Stuttgart possible starting lineup:
Bredlow; Mavropanos, Anton, Ito; Vagnoman, Karazor, Endo, Sosa; Mvumpa, Fuhrich; Guirassy
Hamburger SV possible starting lineup:
Fernandes; Heyer, David, Schonlau, Muheim; Reis, Meffert, Benes; Konigsorffer, Glatzel, Dompe
We say: Stuttgart 2-1 Hamburger SV
A closely-fought contest between two giants of German football, who are both three-time Bundesliga winners, is set to take place on Thursday, although Stuttgart will be regarded as slight favourites.
As the two teams look to quickly brush aside the disappointment of failing to meet their desired targets last weekend, we believe that the hosts will come out on top to gain a first-leg advantage.
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