Background The Silk Road connected the East and West for over 1500 years. Countries in Central Asia are valuable in addressing the hypothesis that parasites on domestic animals were introduced along the Silk Road. Adult fleas are obligate parasites, having worldwide distribution. In dogs, Ctenocephalides canis, C. felis and C. orientis are the most common species identified. The distribution of the Oriental cat flea, C. orientis, is restricted to southeast Asia. The purpose of this study was to determine the diversity of dog fleas from Uzbekistan, a country in Central Asia, with particular reference to C. orientis. Methods Fleas were collected from 77 dogs from 5 locations in Uzbekistan. The cox1 gene sequences from Ctenocephalides spp. were compared to global collection of Ctenocephalides cox1 haplotypes. Landmark-based geometric morphometrics have been applied to the head and curvature to compare C. canis and C. canis using canonical variate analysis and discriminant function analysis. Results Overall, 199 fleas were collected and identified as C. canis (n = 115, 58%), C. orientis (n = 53, 27%) and Pulex irritans (n = 22, 11%). None of the fleas were C. felis. All Ctenocephalides spp. fleas were subject to cox1 amplification and 95% (166/175) yielded DNA sequence. There were 25 cox1 haplotypes; 14 (22/25, 88%) were C. canis cox1 haplotypes and 3 (3/25, 12%) were C. orientis cox1 haplotypes. Molecular analysis confirmed the absence of C. felis. Four (4/22) and one (1/3) cox1 haplotypes were identical to cox1 haplotypes belonging to C. canis and C. orientis cox1 haplotypes identified elsewhere, respectively. Overall morphometric analysis confirmed significant differences between the head shape of C. canis and C. orientis and improved four–fivefold the species identification compared to traditional morphological key. Conclusion We report for the first time the presence of C. orientis in Uzbekistan. Differentiation of C. orientis from C. canis and C. felis remains difficult in regions where these species coexist. Studies in Central and Southeast Asia should confirm species identity using cox1 locus to enable retracing of the distribution of the Ctenocephalides in Asia. The presence of C. orientis suggests that this species may have been introduced from the east along the ancient Silk Road. Graphical Abstract