Exserohilum turcicum is a devastating fungal pathogen that infects both maize and sorghum, leading to severe leaf diseases of the two crops. According to host specificity, pathogenic isolates of E. turcicum are divided into two formae speciales, namely E. turcicum f. sp. zeae and E. turcicum f. sp. sorghi. To date, the molecular mechanism underlying the host specificity of E. turcicum is marginally known. In this study, the whole genomes of 60 E. turcicum isolates collected from both maize and sorghum were resequenced, which enabled identification of 233,022 single nucleotide polymorphisms (SNPs) in total. Phylogenetic analysis indicates that all isolates are clustered into four genetic groups that have a close relationship with host source. This observation is validated by the result of principal component analysis. Analysis of population structure reveals that there is obvious genetic differentiation between two populations from maize and sorghum. Further analysis shows that 5,431 SNPs, including 612 nonsynonymous SNPs, are completely co-segregated with host source. These nonsynonymous SNPs are located in 539 genes, among which 18 genes are predicted to encode secretory proteins, including six putative effector genes named SIX13-like, Ecp6, GH12, GH28-1, GH28-2, and CHP1. Sequence polymorphism analysis reveals various numbers of SNPs in the coding regions of these genes. These findings provide new insights into the molecular basis of host specificity in E. turcicum.