Using mitochondrial DNA (mtDNA) variations, the phylogenetic position of Croatian populations of spined loaches within the European genus Cobitis was assessed. Spined loaches from the Matica, Neretva, Cetina, Zrmanja, Jadova, Sava, Drava and Kupa Rivers are included in two previously described groups: a) Cobitis sensu stricto group, and b) Adriatic group. The Danubian populations of C. elongatoides and its hybrids from the Kupa and Drava Rivers are related to the species included in the Cobitis s. str. group, whereas all other analysed populations clustered within the Adriatic group, which is divided into the “Bilineata”, “Elongata” and “Ohridana-zanandreai” clades. The Croatian spined loaches from the Adriatic watershed are included in the first two clades. Four mitochondrial lineages were revealed within the “Bilineata” clade: 1. ”C. bilineata” lineage containing loaches from the Zrmanja River and Italian and Spanish representatives of C. bilineata; 2. “Neretva-Cetina” lineage included loaches from the Cetina River (C. dalmatina) and the Neretva River (C. narentana); 3. “Jadova” lineage included specimens from the Jadova River; and 4. “Matica” lineage comprised spined loaches from the Matica River. Cobitis elongata from the Kupa and Drava Rivers belonged to the “Elongata” clade with two sublineages. Allozyme analyses of the C. taenia complex revealed the presence of both C. elongatoides and its all-female triploid hybrids in the Danube basin of Croatia.