Spot blotch, caused by the fungal pathogen Cochliobolus sativus, is a limiting factor for barley (Hordeum vulgare) production in northeast China, which causes significant grain yield losses and kernel quality degradation. It is critical to determine the virulence diversity of C. sativus populations for barley resistance breeding and the judicious grouping of available resistance varieties according to the predominant pathotypes in disease epidemic regions. With little information on the barley pathogen in China, this study selected 12 typical barley genotypes to differentiate the pathotypes of C. sativus isolates collected in China. Seventy-one isolates were grouped into 19 Chinese pathotypes based on infection responses. Seventeen isolates were classified as pathotype 3, which has only been identified in China, whereas most (52 of 71) were classified as pathotype 1. All of the tested isolates had low virulence on the North Dakota (ND) durable, resistant line ND B112. Using 22 selected amplified fragment-length polymorphism (AFLP) primer combinations, genetic polymorphism was used to analyze 68 isolates, which clustered into three distinct groups using the unweighted pair group method average with the genetic distance coefficient. No relationship was found between the virulence of isolates and their origins. Isolates of the same pathotype or those collected from the same location did not group into clusters based on the AFLP analysis.