Acidovorax avenae subsp. avenae (Aaa) is a bacterial pathogen associated with the etiolation and decline of creeping bentgrass (Agrostis stolonifera L.) in the United States.  Bacterial diseases pose unique problems in managed turfgrass systems due to their rarity, unconventional infection, and lack of products labeled for their control.  Understanding the emergence of a new disease and developing prophylactic measures often depends on the ability to characterize bacterial pathogen variants.  The genus Acidovorax contains several pathogenic species and subspecies of different monocotyledonous and dicotyledonous hosts.  Many of these subspecies are considered identical, however preferentially attack unique hosts.  Multi-locus sequence analysis is a highly reproducible, portable, typing tool that can more accurately discern among closely related bacterial strains than traditional DNA-DNA reassociation and fragment length gel electrophoresis assays.  The focus of this study is to determine the genetic relationships among the recently isolated strains of Aaa from diseased creeping bentgrass to the more widely studied Acidovorax spp. originating on different hosts, which are largely indistinguishable with conventional 16S rDNA sequence analysis.  A previously developed Multilocus sequence analysis (MLSA) scheme was employed using seven conserved loci (gltA, gmc, ugpB, pilT, lepA, trpB, and phaC) on 28 turfgrass strains and 112 isolates from worldwide collections of pathogenic Acidovorax strains from various hosts; a total of 980 sequences.  Neighbor-joining, maximum-liklihood, and maximum-parsimony phylogenies were compared using concatenated sequences of all seven genes. Topologies rendered three groupings of turfgrass Aaa isolates, distinct from Aaa isolates originating from maize, rice and vasseygrass.  This observed divergence among isolates originating from diseased creeping bentgrass has lead to whole genome sequencing of select strains for further investigations into pathogenicity determinants and development of highly specific molecular diagnostic tools.