Plant trichomes are related to adaptation to stressful environments. However, the mechanisms underlying the association and the genetic basis of plant trichome development are still far from clear. In this study, a dominant gene HAIRY LEAF 4 (HAIL4) controlling wheat leaf trichome production was identified from the Gansu landrace Yizhimai using BSA-Chip approach. The genetic effect of HAIL4 was validated by using three HAIL4 NILs in the backgrounds of Nanda 2419, Huaimai 33 and Yangmai 20, respectively. HAIL4 was fine mapped using recombinants to a 0.085 cM interval that covered two genes TaHisk and TaTRR. TaHisk did not express in both parents, and showed no difference in genomic sequences. TaTRR had a SNP in the fifth exon encoding part of MYB domain, co-segregating with leaf hairiness in a haplotype analysis. Two independent EMS-induced TaTRR mutants derived from Yizhimai confirmed its involvement in leaf trichome development.