Cytological analysis of four Serapias L. (Orchidaceae), of which two are Sicilian endemics, is carried out. In particular, the endemic S. intermedia subsp. hyblaea shows a triploid chromosome complement with 2n = 3x = 54, representing natural nothotaxa arising from a hybridization process between supposed parental S. vomeracea (2n = 2x = 36) and Serapias lingua (2n = 4x = 72). For each of them the C-heterochromatin distribution, using Giemsa C-banding and karyotypes, was examined. The other endemic taxon (S. orientalis subsp. siciliensis) is characterized by a diploid chromosome number 2n = 2x = 36, of which the C-heterochromatin distribution is examined as well. The taxonomical relationships among these taxa are discussed in relation to the literature data.