Technical Abstract: The wheat stripe rust pathogen, Puccinia striiformis Westend. f. sp. tritici Eriks. (Pst), occurred on several experimental wheat (Triticum aestivum L.) lines planted at the North Florida Research and Education Center in Quincy, Gadsden Co., FL in early February 2003. By April, susceptible lines had developed rust severities ranging from 80 to 100% with infection on leaves and leaf sheaths. Stripe rust was prevalent throughout the plots, indicating a uniform rust distribution. Pst-infected leaf samples were analyzed for virulence factors and race identification on a set of wheat genotypes currently used to differentiate races of Pst in North America. An isolate collected from the wheat line FL8524-X7-V1-21-A4-B1-C was identified as race PST-78 (virulence on Lemhi, Heines VII, Lee, Fielder, Express, Yr8, Yr9, Clement, and Compare). The isolate from wheat line FLK-R-7-1-GH-B27-C3 was identified as PST-80 with virulence on the same differential genotypes as PST-78 plus Produra. An isolate from wheat line NC98-26143 was identified as race PST-97 with virulence on the same differential genotypes as PST-78 plus Stephens. An isolate from wheat line 90192-W14-Y1-A7-C9 was identified as PST-98, which has virulence on the same differential genotypes as PST-80 plus Stephens. This report constitutes the first documented case of stripe rust of wheat in Florida.