Aims: To characterize a novel pseudomonad isolate capable of causing brown blotch disease of Agaricus bisporus. Methods and Results: Using the white-line-in-agar (WLA) assay, fluorescent pseudomonads isolated from a New Zealand mushroom farm were screened for the lipodepsipeptide tolaasin, a characteristic marker of Pseudomonas tolaasii. One isolate, NZ17, produced a positive WLA assay and caused brown lesions of A. bisporus comparable with those produced by Ps. tolaasii. However, genetic analysis suggested that Ps. tolaasii and NZ17 were genetically dissimilar, and that NZ17 is closely related to Pseudomonas syringae. Nucleotide sequence analyses of a gene involved in tolaasin production indicated that similar genes are present in both NZ17 and Ps. tolaasii. Conclusions: NZ17 represents a novel Pseudomonas species capable of causing brown blotch disease of A. bisporus. Significance and Impact of the Study: Phenotypic identification of Ps. tolaasii based on A. bisporus browning and positive WLA may have limited specificity.