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Pupilla pratensis (Clessin, 1871) was recently confirmed as a distinct species based on morphological, ecological and molecular evidence. The main purpose of this study is to publish the first reliable data on the occurrence of P. pratensis in the Czech Republic and Slovakia. The second goal is to analyse conchometry of P. pratensis, P. muscorum (L., 1758), and P. alpicola (Charpentier, 1837) to find out whether it is possible to reliably distinguish these species solely based on shell measurements. For multidimensional analysis of shell measurement variation we used principal components analysis (PCA). We documented six populations of P. pratensis in the Czech Republic and one in SW Slovakia. The revision of voucher material showed that all previously reported records of P. alpicola from the Czech Republic belonged in fact to P. pratensis. This requires the exclusion of P. alpicola from the list of Czech molluscs. Based on multidimensional analysis of shell measurements it was possible to distinguish P. pratensis from P. muscorum with no overlapping specimens. Pupilla alpicola was almost completely different from P. muscorum with only few overlapping specimens, contrary to P. pratensis which was mostly impossible to distinguish from P. alpicola based on analysed shell measurements. Shell width was the best single shell measurement for distinguishing P. pratensis and P. muscorum. Shell measurements of two Swedish populations of P. pratensis did not differ from shell variation of Czech and Slovak populations. However, Scandinavian populations displayed some differences from central European populations in apertural barriers which are discussed in detail. Czech and Slovak populations of P. pratensis occurred in calcium-rich fen meadows which perfectly matches with site characteristics reported from Scandinavia. We assume that the observed morphometric differences between P. pratensis and P. muscorum can be useful for distinguishing the species also outside the Czech territory and for palaeoecological studies where only empty shells are available. Since these species occupy ecologically different habitats their reliable identification in fossil material can improve the reconstructions of past environments.