Technical Abstract: Puccinia hordei is the causal agent of barley leaf rust. To study the genetic diversity in P. hordei, 45 isolates with diverse virulence patterns and geographical origins were analyzed using amplified fragment length polymorphism markers. Two pathotypes of Puccinia graminis f. sp. tritici and one isolate of P. graminis f. sp. secalis were included in the analysis for comparison. Six primer-pair combinations of amplified fragment length polymorphism were used and a total of 782 polymorphic markers were generated. Cluster analysis showed that P. graminis f. sp. tritici and P. graminis f. sp. secalis were distinctly different from P. hordei. The P. hordei isolates were clustered into five groups: group I contained a single, rare isolate that was virulent on all resistance genes except Rph13 and Rph15; group II contained a single isolate found to be virulent on the resistance gene Rph15; group III contained 2 isolates; group IV contained 24 isolates, 11 from the United States and 13 from diverse locations around the world; and group V contained 17 isolates, 7 from California, 7 from other states of United States, and 3 from central Europe. The study revealed that molecular diversity in P. hordei can be associated with virulence, but not well with geographic origin.