It is widely acknowledged that understanding of science is key to becoming global citizens and to embrace technological advancements. Although research suggests that girl's performance in science has improved over the years, there are still concerns about the under-representation of women in science, technology, engineering and mathematics (STEM) careers in most African countries, including South Africa. Variability in science performance according to gender is still an unresolved issue. In this study we aimed to examine the relationship between gender and science item achievement of Grade 9 South African learners in the Trends in International Mathematics and Science Study (TIMSS) 2015. We used data collected by TIMSS 2015. The data were further analysed using International Database (IDB) analyser and t-test statistics. The results show that in general gender did not influence learner achievement in most of the test items. However, the findings indicate that girls performed better than boys in most of the questions in all 4 content domains and in the knowing and reasoning cognitive domains. The study highlighted that gender had a limited effect in the manner in which the girls and boys answered the different test items. The observed gender differences in the content domains could be related to socio-cultural environments and learning experiences.