Phylogenetic relationships among six isolates of Halicephalobus gingivalis (Stefanski, 1954), a species with pathogenic potential in horses and humans, were evaluated using DNA sequences from the nuclear large-subunit ribosomal RNA (LSU rDNA) gene. Sequences from nematodes obtained from in vitro cultures (soil or clinical sources), or isolated from infected horse tissues, were compared. Gene sequences from a fatal equine clinical case from southern California and a free-living isolate recovered from southern California soil showed no fixed differences. Sequences from isolates representing two fatal equine cases from North America, one from Ontario, Canada and another from Tennessee also showed no fixed differences. In contrast, two equine cases from Tennessee had 18 fixed differences for this LSU region, the greatest observed among isolates from horses. Phylogenetic analysis of six Halicephalobus sequences and four outgroup taxa by maximum parsimony yielded one tree with five well-supported clades. This phylogeny did not group isolates of Halicephalobus strictly by region of geographic isolation or source of sample, and depicted one clinical and one soil isolate as sister taxa. These results confirm that free-living environmental isolates are potential sources of infection for horses. The phylogeny also reveals that diverse isolates can cause infections in horses within a relatively limited geographic region, and conversely that genetically similar sister taxa can be recovered from geographically distant localities. PCR primers that selectively amplify Halicephalobus DNA were designed and tested based on comparison of closely related nematodes as inferred from phylogenetic analysis.