Educational and psychological research often involves comparing motivation across groups. It is critical to ensure that observed differences in motivation are true variations by group, not due to measurement biases. With a diverse sample of undergraduate students (N= 2200), this study measured internal consistency and gathered validity evidence based on the internal structure of five motivation scales. To compare motivation for biology between groups of undergraduate students, this study tested for measurement scalar invariance by group and, accordingly, conducted latent factor mean comparisons to understand true group differences. On average, female students held lower expectancy beliefs and self-efficacy for biology learning than males. Female students perceived higher attainment value and utility value for biology learning and higher psychological cost. First-generation college students held lower expectancy beliefs and self-efficacy but perceived higher attainment value for biology learning than continuing-generation students. No differences in average motivation for biology learning were found between underrepresented racial minority (URM) and non-URM students. The implications of these findings and future research directions are also discussed.