Since the 1960s, isoenzymes have been well known as one of the most common biochemical markers. Establishing the overall variability of the isoenzyme systems and identifying their genetic control retain their relevance, allowing researchers to reveal the fine mechanisms of the relationship of the organism with the environment and homeostasis and to develop effective biochemical markers for rapid assessment of genetically and selectively significant material. In this paper, the chromosome loci responsible for the activity of 13 different esterase forms of mature seeds in Brassica rapa L. were identified and mapped for the first time. The doubled haploid lines of two mapping populations, DH30 and DH38, were studied. All identified esterase isoforms were divided into three groups according to their electrophoretic mobility. The group of isoforms A1-A3 had a high molecular weight and low electrophoretic mobility. The group of B1-B7 isoforms exhibited an average molecular weight and an average electrophoretic mobility. The C1-C3 group consisted of isoforms having a low molecular weight and, consequently, the highest electrophoretic mobility. Each of the parental forms, as well as each of the studied lines of mapping populations, had its own unique electrophoretic spectrum of esterase isoforms. Based on the electrophoretic data obtained for both populations, a QTL analysis was carried out and chromosome loci were identified, determining the manifestation of each esterase isoform identified in the mapping lines of populations DH30 and DH38. The composite interval mapping approach, combined with a permutation test (1000 iteration) and a confidence level of p < 0.05, allowed us to identify and locate QTLs on chromosomes that determine the manifestation of all esterase isoforms identified by gel electrophoresis, with the exception of A1 isoform for the DH38 population and B7 isoform for the DH30 population. For these two isoforms, no QTL analysis results were obtained because of limitation in initial data on these isoforms in the corresponding mapping population. A total of 35 QTLs for esterase isoforms were mapped for DH30 mapping population and 39 QTLs for DH38 population. As a result of the QTL analysis, molecular markers genetically linked to the identified loci and the percentage of phenotypic variability determined by each of the identified QTLs were also identified. According to isoenzyme analysis, the heterozygosity of both populations in each H_l locus and total heterozygosity H_total, as well as Var(H_l) heterozygosity dispersions for one locus and the variance of average heterozygosity within each population Var(H_total) were calculated. The identified heterozygosity was considered as the average portion of loci with two different alleles in one locus in one individual and could be defined as the observed heterozygosity characterizing the part of the genes for which the studied population is heterozygous. It was shown that in the studied populations of doubled QTL haploid lines, which determine the complex of esterases isoenzymes, are found mainly in the 2nd, 4th, 6th, and 9th linkage groups and form blocks of co-adapted genes and genomic co-adapted gene blocks, which emphasizes the importance of the contribution of these loci in the ontogenesis and adaptability of plants B. rapa. In general, the carried out molecular genetic mapping and biochemical analysis of the studied biochemical traits of various manifestation of esterase isoforms in mature seeds of B. rapa revealed genetic determinants of the studied characters, as well as the genome distribution of mapped QTLs, which in the long term makes it possible to conduct effective molecular and genetic screening of collection accessions and breeding material of the B. rapa species according to these biochemical characters when performing genetic and selection investigations in this species.