Pseudomonas threatens fluid milk and uncultured cheese quality by producing unwanted odors, flavors, and pigments. Pseudomonas likely persists in the dairy plant environment and enters products predominantly as an uncontrolled postpasteurization contaminant. Previous techniques for tracking Pseudomonas in dairy products and plant environments included ribotyping, pulsed-field gel electrophoresis, and RAPD PCR, but these techniques are complex and provide limited and possibly unreliable information on bacterial phylogeny and identity. 16S rDNA sequencing, often used to identify bacteria, lacks the resolution to differentiate between Pseudomonas strains and even species. Our goal was to identify a single housekeeping gene that could be analyzed in dairy spoilage-associated Pseudomonas for accurate species-level identification, subtyping, and tracking of persistent spoilage issues. Initially, 115 Pseudomonas isolates from pasteurized milk produced at 9 different plants were characterized by a 7-gene MLST scheme previously designed for P. fluorescens. This initial data set was used to select a single gene that could be used for simple and affordable Pseudomonas characterization. Based on the number of isolates successfully sequenced, the number of unique allelic types, nucleotide and sequence diversity measures, and measures of recombination, the gene encoding isoleucyl tRNA synthetase (ileS) was identified as the most appropriate. Using type strain Pseudomonas whole-genome sequences, we demonstrated that ileS agrees closely with whole-genome phylogenies and is therefore suitable for isolate identification. Analysis of 91 isolates with the same 16S sequence type from 8 different plants found 28 unique ileS allelic types, greatly improved resolution. Proteolysis and lipolysis screening of 143 isolates showed that ileS clades differed regarding these spoilage-related phenotypes. Overall, our data suggest that ileS sequencing provides a promising strategy for affordable initial characterization of Pseudomonas isolates, which will help the dairy industry in the identification, characterization, and tracking of Pseudomonas in their facilities and products.