Brown blotch disease of white button mushrooms (Agaricus bisporus) is one of the most important and devastating diseases in mushroom cultivation centers. Symptoms of the disease appear as irregular, yellow or brown to black spots on the cap and stem of mushroom. Pseudomonas tolaasii has been cited as the main causal agent of bacterial blotch, but in scientific resources, usually more than one microorganism is reported as the causal agent of brown blotch. In order to investigate the incidence of this disease and also, to identify its causal agent in Shahrekord County, during summer and winter of 2017, mushroom cultivation centers in different cities of Shahrekord County including Shahrekord, Choobin, Vardanjan and…. were visited. Mushrooms with decayed and brown tissues were collected. Bacterial isolation was performed on nutrient agar medium. All kinds of colonies appeared on the plate were purified. The pathogenicity test of the isolates was performed on the blocks prepared from the cap and stem of mushroom and also on intact cap. For identification of isolates, different phenotypic and biochemical tests including gram reaction, growth under aerobic/ anaerobic conditions, oxidase, catalase, arginine dihydrolyase, levan production, potato soft rot, hypersensitive reaction on geranium and utilization of carbon sources were performed. In pathogenicity test, the isolates showed different degrees of browning or blackening and tissue decaying. According the results of phenotypic and biochemical test for example, negative reaction on gram staining, aerobic growth, producing fluorescent pigment on King’s B medium, positive reaction on oxidase and arginine dihydrolyase tests, sensitivity to gentamycin (10 μg/disk) and 4% NaCl tolerance, most of isolates were identified as P. tolaasii. These isolates were also detected in polymerase chain reaction with specific primers pt-1A/1D, so their identification as P. tolaasii was confirmed. The noticeable point was the diversity of isolates that were identified as P. tolaasii based on some phenotypic characters and diseases severity. In pathogenicity tests, some of these isolates showed higher pathogenicity and leaded to faster and further tissue color change on mushroom blocks and also on intact cap. These isolates were also different from others in some phenotypic characters including color and morphology of colony, HR on geranium and potato rot test. It should be mentioned that some of pathogenic isolates were identified as Pantoea sp. based on phenotypic and biochemical characters. Subsequent tests are in progress to identify the species of Pantoea. Based on author’s knowledge, this is the first study for identification of causal agent of mushroom bacterial blotch in Shahrekord County.