Mayflies are one of the most important orders of freshwater inhabiting insects and they are widely used in ecological studies. Mayfly ecology and distribution in the Mediterranean part of the Western Balkans are insufficiently investigated. Our study encompassed 9 sampling sites along the course of the Cetina River, the longest Mediterrranean river in Croatia, and 2 sites in the spring reach of its tributary, Ruda River. Mayflies were sampled monthly between August 2004 and August 2005. A total of 22 mayfly taxa were recorded, including 12 rare species. Due to the variety of available microhabitats and suitable physico-chemical water properties (e.g. water temperature), the highest mayfly richness was recorded in the lower reaches of the river. We discuss distribution and ecology of collected species, particularly the most abundant species (e.g. Baetis rhodani (Pictet, 1843) and Serratella ignita (Poda, 1761)) and the rare species (e.g. Siphlonurus croaticus Ulmer, 1920). One of the rare species collected, Caenis beskidensis Sowa, 1973, has a central and western European distribution and was hitherto not recorded in the Dinaric Western Balkan ecoregion. Morphologically variable specimens from the Rhithrogena group semicolorata were collected, and variability of the most important morphological characters is presented here. In order to resolve the taxonomical status we sequenced the mitochondrial cytochrome oxidase c subunit I (barcode region) of morphologically distinct specimens from the Cetina spring and another locality belonging to the Black Sea drainage (Bijela rijeka, Plitvice Lakes NP). Uncorrected p-distances within and between populations enabled us to associate all specimens to Rhithrogena braaschi Jacob, 1974. Moreover, in contrast to high morphological variability observed in this species, we infer low genetic differentiation of the mtCOI fragment in this species in the Dinaric Western Balkans.