In a modern yet demanding society, scientific literacy (SL) is an essential skill that enables the individual to explain, understand and discuss issues related to science, health, and the environment. The purpose of this research study is to validate the Scientific Literacy Assessment (SLA) tool in the Greek language and investigate the level of SL of 362 Greek pre-service primary school teachers. Reliability and validity were examined using exploratory factor analysis, confirmatory factor analysis and the Cronbach's alpha coefficient, and a statistical analysis was performed to verify the factor structure of the two components of SLA. The results revealed that the level of demonstrated knowledge (SLA-D1) was moderate while motivational beliefs about learning science were satisfactory (SLA-MB). In terms of demographic factors such as gender, high school course specialization and undergraduate year, the results demonstrated an effect on SLA scores. Recommendations for further research in primary teacher preparation programs are presented.