Vertebrate species knowledge is one of the predictors of pupils' understanding of biodiversity. This study will describe vertebrate species identification skills of pupils from the Czech Republic. Altogether, the research tool included 30 vertebrate species, out of which five were fish, three were amphibians, three were reptiles, nine were birds, and 10 were mammals. The research tool consisted of 22 pictures, three footprints, two silhouettes, and three sounds. In addition, we evaluated the influence of variable factors on vertebrate species knowledge, which the research tool also contained. The paper will describe the percentage success rate of vertebrate species knowledge of 1537 respondents. On average pupils could identify nearly 15 species. The results showed that differences in species knowledge were statistically significant mostly by pupils' expectations (self-efficacy) or their results and educational level. In general, younger students identified animals worse than students of higher levels of education. Moreover, significant differences were confirmed between the five classes of vertebrates. Mammals were the best-identified class, followed by amphibians and fish; reptiles and birds were the least correctly identified. While educational level played a significant role in identification skills, the results revealed that the pupils' hometown did not play a significant role.