Non-traditional students represent an important group of university students, and that is why their motivation to study is an important factor that affects current university education. This study investigates the academic motivation of Czech students who are considered non-traditional because of their age (they are older than 26) and at the same time have experienced a break of at least one year in their formal educational trajectory. The Czech version of the Academic Motivation Scale (AMS) has been used to measure academic motivation. The purpose of this study is to examine the factor structure of the Czech version of the AMS on a sample of 1,885 first-year students at Masaryk University and determine if this tool is functional even on a specific group of non-traditional students and to identify differences in particular types of academic motivation between traditional and non-traditional students. The results of confirmatory factor analysis showed that the Czech version of the AMS is a valid scale with a factor structure corresponding to the original model, and based on measurement invariance analysis we can state that the Czech version of the AMS can be used to compare traditional and non-traditional students. The results of regression analyses suggest that non-traditional students had significantly higher values for all types of intrinsic motivation and lower values for most types of extrinsic motivation. In the case of amotivation, it was again the non-traditional students with significantly lower values, which suggests that the absence of a motivation to study tends to be more common in younger students who are continuously receiving formal education.