Hydroxysteroid dehydrogenase (HSDs) is an oil-body sterol protein (steroleosin) with an NADP(H) binding domain that belongs to the short-chain dehydrogenase/reductase (SDR) superfamily. There are numerous studies on the characterization ofHSDsin plants. However, thus far, the evolutionary differentiation and divergence analysis of these genes remain to be explored. The current study used an integrated method to elucidate the sequential evolution ofHSDsin 64 sequenced plant genomes. Analyses were conducted on their origins, distribution, duplication, evolutionary paths, domain functions, motif composition, properties, and cis-elements. Results indicate that except for algae, HSD1 was widely distributed in plant species ranging from lower to higher plants, while HSD5 was restricted to terrestrial plants, and HSD2 was identified in fewer monocots and several dicot plants. Phylogenetic analysis of HSD proteins revealed that monocotyledonous HSD1 in moss and ferns appeared closest to the outgroup,V. carteriHSD-like,M. musculusHSD1, andH. sapiensHSD1. These data support the hypothesis thatHSD1originated in bryophytes and then in non-vascular and vascular plants, followed byHSD5only in land plants. Gene structure analysis suggests thatHSDsin plant species came up with a fixed number of six exons, and the intron phase was primarily 0, 1, 0, 0, and 0. Similarly, duplication analysis revealed that segmental duplications were the main reason forHSDsin plant species. Physicochemical properties suggest that dicotyledonous HSD1s and HSD5s were mainly acidic. The monocotyledonous HSD1s and HSD2s and the dicotyledonous HSD2s, HSD3s, HSD4s, and HSD6s were mainly basic, implying thatHSDsin plants may have a variety of functions.Cis-regulatory elements and expression analysis revealed thatHSDsin plants might have roles in several abiotic stresses. Due to the high expression ofHSD1sandHSD5sin seeds, theseHSDsin plants may have roles in fatty acid accumulation and degradation.