Leaf pubescence is widespread among higher plants. In bread wheat, a relationship was found between this trait and the efficiency of photosynthetic processes and productivity. In this work, we established the chromosomal localization of the gene for leaf pubescence introgressed from Triticum timopheevii into a bread wheat line 821 and studied its expression in the genetic background of two wheat cultivars differing in genetic control and phenotypic expression of pubescence. To obtain quantitative characteristics of pubescence in cultivars and hybrid populations, the LHDetect2 program was used, which makes it possible to estimate the length and number of trichomes on a leaf fold. A genetic analysis showed the dominant inheritance of the gene. Monosomic analysis F2 was used to establish chromosome localization and investigate the expression of the gene in cultivars Saratovskaya S29 (S29) and Diamant 2 (Dm2). As a result, the gene Hltt, introgressed from T. timopheevii, was identified and localized in the distal region of the long arm of 5A chromosome for the first time. In both F2 populations, the gene reduced the density of trichomes and formed long trichomes, uncharacteristic for the two recipient cultivars S29 and Dm2. A larger number of long trichomes was formed in the genetic background of S29, which carry the bread wheat gene Hl1 and Hl3 for leaf pubescence, than in Dm2. Development of substitution and isogenic lines with the fragment of introgression carrying the gene Hltt will allow determining function and assessing the adaptive significance of the gene more precisely.