Knowing the molecular mechanism of male sterility in alfalfa is important to utilize the heterosis more effectively. However, the molecular mechanisms of male sterility in alfalfa are still unclear. In this study, the bulked segregant analysis (BSA) and bulked segregant RNA-seq (BSR) were performed with F2 separation progeny to study the molecular mechanism of male sterility in alfalfa. The BSA-seq analysis was located in a candidate region on chromosome 5 containing 626 candidate genes which were associated with male sterility in alfalfa, while the BSR-seq analysis filtered seven candidate DEGs related to male sterility, and these candidate genes including EF-Tu, β-GAL, CESA, PHGDH, and JMT. The conjunctive analyses of BSR and BSA methods revealed that the genes of Msβ-GAL and MsJMT are the common detected candidate genes involved in male sterility in alfalfa. Our research provides a theory basis for further study of the molecular mechanism of male sterility in alfalfa and significant information for the genetic breeding of Medicago sativa.