Esterases represent a large group of enzymes that catalyze cleavage of multiple-ester bonds. In general, they are divided into four types: cholinesterases (most frequently, these are identified using ordinary electrophoretic analysis), acetylesterases, arylesterases and carboxyl esterases. Plant carboxyl esterases catalyze conversion of the esters into bioactive acids and alcohols, thereby playing a key role in many biological processes. Lack of epistatic interactions as well as a co-dominant nature of the inheritance of the esterase isozymes makes them meaningful for quick and accessible investigation of the processes of biochemical adaptation to environmental changes. Such a type of markers, which is convenient for solution of practical problems of selection, can be used as a tool that can speed up and simplify the selection of the significant material. The aim of this study was to estimate the isoenzyme profile of esterases isolated from mature seeds, and to ascertain, using such a biochemical marker, the polymorphism among samples of promising breeding material of the hexaploid wheat (Triticum aestivum L.). Ripe seeds from following wheat cultivars were used as samples: Zlata, Lyubava, Agatha, Lisa (spring wheat) and Mera (winter wheat) (originated by Moscow Agricultural Research Institute «Nemchinovka», Moscow Province); lines AFI91 and AFI177 (spring wheat) and recombinant inbred lines of the mapping populations ITMI - 7, 10, 29, 32, 44, 47, 57, 83, 88, 89, and 115 (spring wheat) (originated by Agrophysical Research Institute - AFI, St. Petersburg). The seeds were ground in a porcelain mortar and the flour was sieved. The enzymes were extracted from the flour and subjected to vertical native electrophoresis using 4 % concentrating and 8 % separating polyacrylamide gels. Molecular weight markers were Page Ruler Prestained Protein Ladder «Thermo Scientific» (Lithuania). After electrophoresis, gels were treated with a reagent for a nonspecific esterase and scanned. Individual electrophoretic profile of each sample was estimated. Heterozygosity and its dispersion were calculated. The esterase complex in the wheat seeds studied was represented by 10 isoforms. From 9 to 10 isoforms of various electrophoretic mobility have been identified in cultivars Zlata, Lyubava, Agatha, Lisa and Mera, 7 isoforms were found in line AFI91, 8 isoforms - in AFI177, and from 7 to 10 isoforms - in ITMI lines. All samples were characterized by the presence of esterases isoforms Est-8, Est-9 and Est-10. Heterogeneity was only found in the qualitative and quantitative composition of esterases with a greater molecular weight - Est-1, Est-2, Est-3, Est-4, Est-5, Est-6, and Est-7. Each variety among eighteen varieties and lines had a genotype different from that in the other samples. The average heterozygosity (H) of samples within 10 loci encoding esterase isoforms was 0.924; the dispersion of heterozygosity for all the samples studied was Var(H) = 0.0004. As a result of the analyses, following most promising breeding parental forms are varieties Zlata and Mera, and lines AFI91, AFI177, ITMI7, ITMI44, ITMI83, and ITMI115. Because of the existence of ten isoforms in hexaploid wheat, the esterases might represent a convenient biochemical marker suitable for examination of samples of the hexaploid wheat at physiological, biochemical, and genetic levels.