Chemistry is a school subject that is not viewed favorably among pupils. Before we can improve pupils' attitudes toward chemistry, it is important to find out the problem as to why the attitudes are relatively negative. The research was focused on Czech lower secondary and secondary grammar school pupils’ attitudes to the subject of chemistry. Also, the difference between groups of variables (gender and grade) was examined. The sample size contained 931 Czech lower secondary and secondary grammar school pupils. The research tool was a questionnaire with 25 Likert type items. By the use of factor analysis, the items were distributed into four categories: 1) Popularity and difficulty of chemistry, 2) The relevance of chemistry, 3) Chemical aids and laboratory experiments, 4) The future life and chemistry. The overall score indicated neutral/slightly positive pupils’ attitude toward chemistry. Girls received a lower score in comparison with boys in all grades, except the 1st grade, in secondary grammar schools. The opposite situation was among lower secondary school pupils, where girls achieved a slightly higher attitude score. It is possible to say that negative attitudes toward chemistry are also among lower secondary and secondary grammar school pupils, so it is needed to try to improve attitudes toward chemistry from the early age of pupils. The recommendations for the pedagogical practice are suggested in the conclusion.