Wheat (Triticum aestivum L.) is a major staple crop in East Africa (EA) providing 9% and 10% of daily calories and protein intake, respectively. However, EA countries depend on import to meet 55% of their domestic wheat supplies due to increasing demands and low domestic yields. To determine the beneficial gene pool currently exploited for wheat improvement in EA, we examined the allelic diversity of 39 genes of breeding importance in a collection of 239 wheat cultivars and breeding lines from Kenya and Ethiopia using KASP markers. The assayed genes have been shown to control variations in plant height, thousand kernel weight (TKW), grain protein content, pre-harvest sprouting (PHS), disease resistance, and flowering time. We observed the beneficial alleles of some major genes including Gpc-B1, Yr5, Yr15, Sr26, and Fhb1 to be missing or present at low frequencies in this population. Furthermore, we validated the effects of the major Rht-1 alleles and TaCKX-D1 in controlling variation in plant height and thousand kernel weight, respectively, under EA conditions. Our results uncover hitherto unexploited allelic diversity that can be used to improve the genetic potential of EA wheat germplasm. This will inform strategies to rapidly mobilize these beneficial alleles for wheat improvement in EA.