Background Understanding pre-service science teachers' (PSTs) perceived knowledge on a subject and teaching that subject is important to better prepare these teachers for their future career. Purpose In this study, PSTs' perceived Technological Pedagogical Content Knowledge (TPACK) on genetics and the contribution of the dimensions of perceived TPACK on PSTs' subject matter knowledge of genetics were investigated. Moreover, the differences in the perceived TPACK with regard to gender and grade level were examined further. Sample 1530 PSTs from eight public universities located in Central Anatolia in Turkey participated to the study. Design and methods Perceived TPACK on Genetics Questionnaire (MaKinster, Boone, and Trautmann 2010) and Test of Basic Genetic Concepts (Sadler and Zeidler 2005) were used. Regarding perceived TPACK eight sub-dimensions were emerged, namely; Educational Technology Knowledge (ETK), Genetic Technology Knowledge (GTK), Project Specific Technology Knowledge (PSTK), Content Knowledge (CK), Pedagogical Knowledge (PK), Pedagogical Content Knowledge (PCK), Technological Content Knowledge (TCK), and TPACK. Descriptive analysis, multiple linear regression analysis, and MANOVA analyses were conducted to provide answers to research questions. Design and methods The students were surveyed before and after the program using open-ended and Likert scale items, and were asked to complete a nuclear science version of the Draw-A-Scientist-Test. Results The participants' mean value of total TPACK is 4.15 out of 6. Pre-service science teachers perceived themselves most knowledgeable on PK and least knowledgeable on PSTK dimensions. The content-related perceived TPACK dimensions significantly contributed to PSTs' subject matter knowledge of genetics. The mean scores of male and female PSTs were found significantly different in PSTK, PK, PCK, TCK and TPACK dimension. ETK, GTK, PSTK, and CK scores of participants were also found to be significantly different with regard to grade level. Conclusions This study provided descriptive information about PSTs' levels of TPACK. Moreover, this study also showed the contribution of content-related perceived TPACK dimensions on subject matter knowledge of genetics. This study showed that female PSTs and PSTs, who took courses in which science, technology, and pedagogy are taken as an integrated manner, had better perceived TPACK. We can conclude that PSTs' perceived TPACK knowledge is situated in a particular subject matter area. Therefore, instead of seeking for TPACK in a general domain, focusing on specific subject matter areas can give us more insight into the nature of the TPACK and better development of PSTs' TPACK.