The domestic water buffalo (Bubalus bubalis) is an economically important livestock species in many Asian and Mediterranean countries. Two types of water buffalos are river and swamp buffaloes. Genetically, the swamp buffalo has 48 chromosomes (2n = 48) and the river buffalo has 50 chromosomes (2n = 50). Khuzestan province is one of the important centers of buffalo farming in Iran. Since there was no cytogenetic information about Khuzestani buffalo breed, the present study was performed to provide karyotype and cytogenetic study in Khuzestan buffaloes. In this study, blood samples of 40 buffaloes were collected randomly from jugular vein via heparinized syringes. For metaphase smear, blood samples were cultured in RPMI 1640 medium containing fetal bovine serum and phitohemaglutination and then transferred to water bath for 69 hr. After stopping cell division by colcemide and using fixed solution, from each sample six slides were stained with Giemsa staining. Based on the results, number of chromosomes of the Khuzestani buffaloes was 50 (2n = 50). Among the 25 pairs of chromosomes, the first 5 pairs of autosomal chromosomes were sub-metacentric, 19 pairs of autosomal ones were acrocentric and the sex chromosomes (X and Y) were acrocentric. Furthermore, X and Y chromosomes were the largest and smallest acrocentric ones, respectively. Therefore, the buffaloes of Khuzestan province are considered as a river type.