In recent years, tan spot of wheat caused by the fungus Pyrenophora tritici-repentis has become more prevalent in Oklahoma. Experiments were conducted to investigate the race structure, disease symptoms and genetic variability in P. tritici-repentis isolates collected from winter wheat over three decades. Race determination was conducted for 16 isolates based on expression of necrosis and/or chlorosis produced on wheat differentials. Variability in disease symptoms expressed by 12 isolates was determined on 13 hard red winter wheat cultivars grown in Oklahoma. In addition, genetic variability among 17 isolates was determined using amplified fragment length polymorphism-polymerase chain reaction (AFLP-PCR). All isolates except one (El Reno) were classified as race 1. Isolates varied widely in producing necrosis and/or chlorosis symptoms on wheat cultivars, but necrosis with a chlorotic halo was predominant (56.4%). AFLP-PCR analysis using 13 primer pairs produced a total of 494 alleles of which 285 were polymorphic. The overall genetic diversity among the isolates was 25.2%. Genetic relationships based on cluster analysis and principal component analysis showed only minor differences between isolates, and isolates did not form tight clusters or groups. The isolates of P. tritici-repentis were predominantly race 1; however, they produced a range of tan spot symptoms on wheat cultivars. The lack of distinct genetic grouping by the AFLP marker study indicates that the isolates used in this study likely originated from a single lineage.