Poa annua L. has long been a cultivated weed on golf courses. However, the recent development of improved cultivars of creeping bluegrass (Poa annua reptans Hausskn.) has generated an increasing interest in selection and breeding of this species. Inter-simple sequence repeat (ISSR) PCR is a relatively new method for genotype identification and measuring genetic diversity and was employed in this study for differentiating among creeping bluegrass genotypes. The objectives of this study were to test ISSR primers for production of polymorphic fragments and ascertain the applicability of ISSR PCR to distinguish closely related genotypes. Eight primers produced fragments, of which 77.3% were polymorphic, and primers UBC849, UBC850, and UMN001 produced over 75% of the total polymorphic fragments. These three primers had sufficient resolution to distinguish all but two of the diploid creeping bluegrass accessions. This method was a simple, fast, and relatively inexpensive method to produce useful DNA fragments in creeping bluegrass. It is a robust method for detecting polymorphic loci that can be used in the study of genetic relatedness, heritability, or linkage to important traits, development of linkage maps, and marker-assisted breeding.