Apostichopus japonicus is a species of economic importance cultured in northern China. In the past 10 years, the scale of the A. japonicus culture has expanded due to increased market demand for products. However, several challenges, such as germplasm degradation and the lack of stress-resistant varieties, have emerged in A. japonicus culture. Breeding new varieties is one way to overcome these challenges. New strains (F1 and F4) of A. japonicus with high temperature resistance were obtained by domestication and screening under environmental stress conditions. The genetic changes caused by breeding under environmental stress conditions not only originated from changes in the DNA sequence, but also from changes due to epigenetic modifications by bisulfite sequencing. To explore the genetic diversity of the selected populations (F1 and F4) and the control population (F) which was not subjected to temperature stress of A. japonicus, the genetic diversity of control population, selected population F1, and selected population F4 were analyzed using methylation-sensitive amplification polymorphisms (MSAP). The results showed that 698 loci were polymorphic among the 806 loci obtained by 10 pairs of primers, and the percentage of polymorphism was 86.60%. Based on the genetic analysis of non-methylated loci, the Shannon polymorphism index of the F4 was 0.3981 and Nei gene diversity was 0.2264. Based on the analysis of methylation sensitive sites, the Shannon polymorphism index of the F4 was 0.5873, and Nei gene diversity was 0.2598, both of which were higher than in the parent population. Moreover, the epigenetic diversity was higher than the sequence genetic diversity caused by variation in non-methylated loci, indicating that the frequency of epigenetic variation was higher than that of sequence genetic variation. Analysis of the MSAP methylation patterns revealed some changes in the methylation levels and patterns in the selected F1 and F4 after breeding, which indicated that the genome methylation status of the A. japonicus population was changed by breeding under temperature stress. The number of type Ⅱ bands in the F4 was 161, which was significantly higher than that of control population of A. japonicus and may have acquired epigenetic characteristics during breeding. Collectively, our results revealed the genetic changes and progress of the breeding population from the perspective of a genetic material basis and provide a reference for the study of epigenetics in the breeding of new stress resistant varieties.