Fine mapping of wheat powdery mildew-resistance gene Pm4e to a 0.19 cM interval with sequence-based markers provides the foundation for map-based cloning and marker-assisted selection with breeder-friendly markers. Powdery mildew caused by Blumeria graminis f. sp. tritici is a wheat foliar disease that poses a serious threat to global wheat production. Pm4 is a resistance gene locus that has played a key role in controlling this disease in wheat production and a few resistance alleles of this locus have been identified. We have previously mapped the Pm4e allele to a 6.7 cM interval on chromosome 2AL. In this study, Pm4e was delimited to a 0.19 cM interval flanked by Xwgrc763 and Xwgrc865, through employment of a larger segregating population, derived from the cross of resistant parent D29 with susceptible parent Yangmai 158 (Y158), and enrichment of the genetic interval with markers developed on Chinese Spring (C.S.) survey sequence. In this interval, Pm4e co-segregated with a few markers, some of which were either D29-dominant or Y158-dominant, implying great sequence variation in the interval between D29 and Y158. Most of these co-segregation markers could not differentiate the Pm4 alleles from each other. Survey of 55 wheat cultivars with four co-dominant markers showed that the Pm4e-co-segregating loci always co-exist. Annotation of the Pm4e interval-corresponding C.S. sequence revealed more than a dozen resistance gene analogs clustered in a 2.4 Mb region, although C.S. is susceptible to the Pm4e-avirulent isolate Bgt2. This study has established the foundation for map-based cloning of Pm4e. Moreover, some of the co-dominant markers developed in this study could help in marker-assisted transfer of Pm4e into elite cultivars.