Motivation for biology can be considered as one of the most important factors influencing the teaching and learning of biology. Thus, the main objectives of this research were to test the validity of an instrument that measures students' motivation for the subject of biology, and to determine the situational motivation of Slovenian secondary school students for learning biology. Based on 516 completed web questionnaires filled in by various secondary school students from Slovenia, we came to the following conclusions. By applying non-parametric tests, we concluded that there were certain statistical differences between genders and between students from different secondary schools and regions when it came to motivation for studying biology. First-year students are more concerned with assessing knowledge. Those from the fourth year, on the other hand, are more interested in the knowledge they have acquired rather than the grade they have achieved. They also considered knowledge in biology as important for their life and thus for their career. The factor analysis yielded a structure different from the theoretical constructs. The responses of Slovenian secondary school students formed five factors for the motivation to learn biology: (1) intrinsic motivation and personal importance, (2) anxiety about assessment tasks, (3) self-efficacy in assessments, (4) career motivation and (5) responsibility. This information could be useful for teachers and researchers to promote motivation for the discipline and subject of biology. A lack of motivation in science can hinder science and science literacy, which is necessary for responsible decision-making and behaviour and in choosing a career in science.