Protein kinases of the MAPK cascade family (MAPKKK–MAPKK–MAPK) play an essential role in plant stress response and hormone signal transduction. However, their role in the cold hardiness ofPrunus mume(Mei), a class of ornamental woody plant, remains unclear. In this study, we use bioinformatic approaches to assess and analyze two related protein kinase families, namely, MAP kinases (MPKs) and MAPK kinases (MKKs), in wildP. mumeand its varietyP. mumevar.tortuosa. We identify 11PmMPKand 7PmMKKgenes in the former species and 12PmvMPKand 7PmvMKKgenes in the latter species, and we investigate whether and how these gene families contribute to cold stress responses. Members of theMPKandMKKgene families located on seven and four chromosomes of both species are free of tandem duplication. Four, three, and one segment duplication events are exhibited inPmMPK,PmvMPK,andPmMKK, respectively, suggesting that segment duplications play an essential role in the expansion and evolution ofP. mumeand its gene variety. Moreover, synteny analysis suggests that mostMPKandMKKgenes have similar origins and involved similar evolutionary processes inP. mumeand its variety. A cis-acting regulatory element analysis shows thatMPKandMKKgenes may function inP. mumeand its variety’s development, modulating processes such as light response, anaerobic induction, and abscisic acid response as well as responses to a variety of stresses, such as low temperature and drought. MostPmMPKsandPmMKKsexhibited tissue-specifific expression patterns, as well as time-specific expression patterns that protect them through cold. In a low-temperature treatment experiment with the cold-tolerant cultivarP. mume‘Songchun’ and the cold-sensitive cultivar ‘Lve’, we find that almost allPmMPKandPmMKKgenes, especiallyPmMPK3/5/6/20andPmMKK2/3/6, dramatically respond to cold stress as treatment duration increases. This study introduces the possibility that these family members contribute toP. mume’s cold stress response. Further investigation is warranted to understand the mechanistic functions of MAPK and MAPKK proteins inP. mumedevelopment and response to cold stress.