Anthracnose basal rot (ABR) is a serious disease of turfgrasses that is caused by the pathogen Colletotrichum graminicola. The relationships of isolates causing ABR on turfgrasses to those causing disease on important crop hosts (maize, sorghum) remain unresolved. Genetic variation among isolates from annual bluegrass, creeping bentgrass, maize, and sorghum was evaluated based on host origin and geographic origin. Isozymes were used to estimate the genetic variation of the isolates. Five enzyme systems comprising 16 alleles from 5 loci were used. Allele frequencies, genetic distance, and linkage disequilibrium values were calculated for isolates based on both host and geographic origin. Isolates from creeping bentgrass and annual bluegrass were the most closely related based on Nei's genetic distance, while isolates from maize and sorghum were the most distantly related, consistent with their known species-level relationship. Isolates from annual bluegrass and creeping bentgrass had different genetic distances to isolates from both maize and sorghum. Annual bluegrass isolates from different geographic regions had the smallest genetic distance values observed in this study, indicating a very close relationship regardless of geographic origin. Based on these data, it appears that host origin, not geographic origin, plays a more important role in the genetic diversity of these fungi.