The purpose of the study was to assess the performance of pre-service teachers in three science formative assessment tests in an online learning environment. The participants consisted of 138 pre-service teachers (57 participated in synchronous session and 81 participated in asynchronous sessions). The study used a quasi-experimental design, non-equivalent groups post-test only. Tests were used to collect the data: Energy Concept Test (ECT), Teaching Energy Concept Test (TECT) and Basic Electronics Concept Test (BECT). ANOVA results revealed a significant effect of type of test on test scores of students at the p < 0.05 level for the three tests [F (2, 364) = 9.641, p = 0.000]. For the ECT, there was no significant difference in performance between synchronous group (M = 15.93, SD = 3.098) and asynchronous group (M = 14.75, SD = 3.691), t (1.791) p = 0.076. For the TECT, there was no significant difference in performance between synchronous group (M = 13.55, SD = 4.069) and asynchronous group (M = 13.97, SD = 3.312), t (-0.596) p = 0.553. Also, for the BECT, there was no significant difference between synchronous group (M = 16.53, SD = 2.727) and asynchronous group (M = 15.19, SD = 3.725), t (2.287) p = 0.024. Pre-service teachers’ performance in the three tests was higher than average. Online learning is an effective medium for teaching and learning of science and can be analogous to face-to-face instruction. Both synchronous and asynchronous environments prove effective for teaching and learning science.