Exploitation of host resistance is important for controlling powdery mildew of wheat (Triticum aestivum L.). In this study, a wheat-Thinopyrum intermedium introgression line, designated WE99, conferred seedling resistance to 47 of 49 Blumeria graminis f. sp. tritici isolates. Genetic analysis demonstrated that the resistance segregation deviated significantly from a single gene Mendelan ratio. However, marker analysis indicated that only a single recessive resistance gene, temporarily designated PmWE99, conferred powdery mildew resistance (Pm). PmWE99 was mapped to chromosome arm 2BS and linked to the three simple sequence repeat markers Gwm148, Gwm271, and Barc55. Using race spectrum analysis, PmWE99 was shown to be significantly different from the documented genes Pm42 and MlIW170 located on chromosome arm 2BS and, thus, appeared to be a new Pm gene. Examination of the genotype frequencies in the F 2:3 families showed that a genetic variation in the PmWE99 interval that favored the transmission of the WE99 allele could be the cause of the deviated segregation. Further investigation revealed that the abnormal segregation only occurred at the PmWE99 interval and was not common at other loci in this population. Identification of PmWE99 will increase the diversity of the Pm genes for wheat improvement.