Stripe rust, caused by Puccinia striiformis f.sp. tritici (Pst), is an important disease of winter wheat in Central Asia. Stripe rust races contain diverse virulence/avirulence patterns and change rapidly. Therefore the objectives of this research were to: (i) examine current pathotype variability of Pst races collected from Kazakhstan and Uzbekistan and (ii) evaluate stripe rust resistance in leading cultivars and advanced breeding lines targeted to those regions. Analyses of 152 Pst samples showed diverse virulence patterns with avirulence to Yr5, Yr10 and Yr15 being common. Most of identified races are among the rare. Analysis of a mixed Pst population showed 10 distinct pathotypes with frequencies ranged from 1.2 to 8.7%. The virulence patterns ranged from least ‘31–1.5’ and X-1.5 to highly virulent ‘86 + E16’. Seedling evaluation of 62 genotypes using the 10 pathotypes showed variations for resistance. Bunyodkor and Barhayot showed resistance to all pathotypes. Five Yr genes were postulated. Yr1 in KR12-5075, and Yr6 in KR11-03 and KR12-5003 were postulated. Yr5 combined with Yr10 and Yr15 genes were determined in Bunyodkor. The wheat genotypes also showed different levels of resistance in adult plant stage under field conditions. Twenty genotypes showed <20% severity in both Kazakhstan and Uzbekistan. The disease severity on several genotypes differed in this countries, suggesting different Pst populations in the two countries. Several resistant genotypes were identified, which should be further evaluated for release as new varieties or used in breeding programmes. Two resistant lines from this study were identified as new varieties in Georgia and Uzbekistan.