The karyotypes of five tooth-carps of Iran, Aphanius persicus, Aphanius sophiae, Aphanius dispar, and Aphanius sp. have been investigated by examining metaphase chromosomes spreads obtained from gill epithelial and kidney cells. The diploid chromosome numbers of all five species were 2n=48. The karyotypes consisted of 8 pairs of submetacentric and 16 pairs of subtelocentric chromosomes in A. persicus; 4 submetacentric and 20 subtelocentric in A. sophiae; 7 submetacentric and 17 subtelocentric in Aphanius ginaonis and 8 submetacentric and 16 subtelocentric in A. dispar. It was 16 metacentric, 1 submetacentric and 7 telocentric chromosome pairs in Aphanius sp. specimen. The arm numbers were 32, 28, 31 and 32 in A. persicus, A. sophiae, A. ginaonis and A. dispar, respectively. It was 41 in Aphanius sp. Sex chromosomes were cytologically indistinguishable in these tooth-carps. Cluster analysis revealed the closeness of A. sophiae and A. persicus. According to our data A. ginaonis is related to a group of A. sophiae+A. persicus+A. dispar.