Abstract Genetic diversity is an important resource to improve new wheat cultivars in the breeding program. In this study, fluorescence in situ hybridization (FISH) and genotyping by sequencing (GBS) were used to evaluate the genetic diversity and population structure of the timopheevii group (AtAtGG, 2n = 4x = 28) including 15 Triticum timopheevii Zhuk. accessions and 35 Triticum araraticum Jakubz. accessions. FISH analysis showed that there were 14 and 19 FISH signal variations in the At and G genome of T. araraticum, respectively. But there were only two signal variations in the G genome of T. timopheevii. In this study, 190,402 SNP markers were obtained from GBS, in which the lowest and highest frequencies of SNPs were found in the G and At genomes, respectively. Genetic diversity analysis of the 50 accessions indicated that the mean GD and PIC were 0.30 and 0.26, with the ranges of 0.1–0.5 and 0.1–0.4, respectively. The highest and lowest numbers of SNPs identified on the chromosomes 4G and 2At were 7,748 and 17,527, respectively. Structure and cluster analyses divided 50 accessions into two subpopulations (POP1 and POP2). POP1 mainly consisted of most Triticum araraticum accessions, and POP2 comprised all accessions of Triticum timopheevii and one Triticum araraticum accession AS273. In addition, the genetic variance showed that genetic variation was greater within populations (97%) than between populations (3%). POP1 and POP2 had high levels of genetic diversity and POP1 showed higher genetic diversity. The POP1 (I = 0.53, He = 0.35, uh = 0.35) showed higher genetic diversity than POP2. The present results provided important information for the improvement of cultivated durum and hexaploid wheat production in future breeding programs of China and other countries.