Medical laboratory technicians (MLTs) play an essential role in healthcare and the profession has to deal increasingly with staff shortage. Consequently, retention becomes a crucial issue. So far little attention has been paid to generational differences in factors regarding job satisfactions of MLTs and comparatively few studies in general have included the youngest cohort of Generation Z. The aim of this study was to analyze aspects of MLTs job satisfaction from a generational perspective and to identify potential for improvement responding to the different generations. A cross sectional online survey study design was used. A total of 382 MLTs completed the online survey of which 371 were assigned to one of the three generational cohorts. The analysis focused on the satisfaction with several aspects of job satisfaction, the gap between the relevance and realization if these aspects and the influencing factors for job satisfaction according to the respective generation. The results showed that recognition represents the most important influencing factor for job satisfaction, but other aspects are mainly relevant for one of the three generations, such as relationships for Generation X, meaningful contribution for Generation Y and income for Generation Z. Employers and professional associations can draw on these findings to positively influence job satisfaction, to support this essential healthcare workforce.