Research was done during the period (October 2009 to September 2010), aiming to identify two species of Cobitis (Cobitis taenia and Nemachilus tigris) belonging to Cobitidae and Gambusia affnis by using karyotype examination. Fish specimens were caught alive from the river Orontes drainage and its tributaries in Idlib and Hama departments by routine fishing gears, and submitted Colchcine treatment in Aleppo agricultural research center, General commission for Scientific Agricultural research which included an incubating in Colchicine medium (0.06%) for 2.5 -3 hours. Fishes were chopped and placed in hypotonic 0.4% KCl solution for 30 min. Afterwards, tissues were fixed in fresh Carnoy solution (3 parts methanol: 1 part glacial acetic acid). Cell suspension was dropped onto slides, dried and then stained in 4% Giemsa solution. Chromosome spreads on microscope slides were examined and several metaphase plates were selected and photographed. Chromosomes of fish studied were grouping into series, i. e., meta-submetacentric and subtelocentric-acrocentric elements, and aligned serially from the larger to smaller, karyograms were constructed and chromosomal formulas and the number of arms were established, as following: Cobitis taenia: 2n=50=14m/sm+36t/st (NF=64), Nemachilus tigris: 2n=50=12m/sm+38t/st (NF=62), Gambusia affinis (female): 2n=48=1m+2sm+45t/st(NF=51), Gambusia affinis(males): 2n=48=2m+46t (NF=50(.