<p>Textbooks are a primary source of biological knowledge and play a key role in shaping students’ understanding of biodiversity. We therefore analyzed the occurrence of organisms in text and images of 21 textbooks from six “textbook sets” published over the last 40 years in Slovak Republic (Slovakia), Czech Republic (Czechia) or former Czechoslovakia. The number of organisms in the Slovak textbook sets is similar to that of the former Czechoslovakia textbook set. On the contrary, all Czech textbook sets contained significantly more mentions of organisms in text (almost twice). The main finding is that mainly Czech textbooks contain more animals than plants in text and in images. Such content may stimulate students’ interest in animals at the expense of plants. Surprisingly, no change was observed in the representation of endangered organisms in these textbooks over 40 years. Textbooks in each set contained significantly more endangered animals than plants as well as animal exotic species (not occurring naturally in these states). Although the most recent textbook sets from both countries contain more photographs (77–91% of all images vs. 3–21% in the past), the Slovak textbooks contain more scientific names, but fewer endangered and exotic organisms compared to the Czech ones. Our analyses also indicate a shift from memorization to an emphasis on developing interest in biology and environmental attitudes in recent Czech textbook compared to the Slovak ones, along with more frequent mention of bacteria/viruses in the post-covid era.</p>