Creeping bentgrass (Agrostis stolonifera L., CB) is widely used for putting greens across Missouri.  Physiological stress accrued from maintaining CB in the transition zone during summer months predisposes plants to infections from soil borne phytopathogens.  In 2011, a considerable outbreak of Pythium root rot (PRR) prompted a 3-year survey to determine which Pythium spp. were most prevalent throughout the Midwest region.  Cup-cutter sized samples (~ 930 cm³ volume) were collected from asymptomatic and symptomatic CB putting greens in Arkansas, Illinois, Kansas, Missouri, and Oklahoma.  Symptomatic roots were washed of soil and observed for Pythium spp. presence under stereo and compound microscopy.  Asymptomatic samples were processed by taking a 30 mg root subsample collected 2.54 cm from the soil line.  Roots were washed under continuously flowing tap water for 6-10 h, rinsed twice with sterile distilled water, and sectioned into 5 mm segments.  Thirty segments were incubated at 23°C on each of three culture media types: selective media amended with 10 mg pimaricin, 250 mg ampicillin, 10 mg rifampicin, and 25 mg pentachloronitrobenzene, water agar (WA), and clarified V8 juice cholesterol agar.  Hyphae emerging after 12h, 24h, and each subsequent 24h period up to 120 hours were transferred to WA.  Isolate identification was performed by observing reproductive structure morphology under light microscopy after culturing on Festuca arundinacea leaves.  DNA barcoding of ITS and COX1 gene regions utilizing universal and Oomycete specific primers confirmed morphological identification.  Eighteen Pythium isolates have been identified from 42 samples representing eight Pythium spp. including P. aphanidermatum, P. arrhenomanes, P. graminicola, P. rostratum, P. torulosum, P. ultimum, P. vanterpooli, and P. vexans.  All species collected are known pathogens of CB except P. rostratum, P. torulosum, and P. vexans.  The virulence of collected isolates on CB will be determined with future field and greenhouse experiments.