As the staple food source for more than one half of the world’s population, rice (Oryza sativa L.) is one of the most important crops in the world. Leaf hair derives from the epidermal cell of leaf and has the functions of resistance to biotic and abiotic stresses in rice. Therefore, it is of great importance to identify genetic loci related to leaf hair development and then subsequently isolate corresponding genes in rice. In recent years, genome-wide association study (GWAS) has been adopted to study complex agronomic traits of crops. Here, we selected 149 rice varieties with abundant phenotypic and genetic variations to detect genetic loci related to development of leaf hairs with GWAS. Total of 13 genetic loci were found significantly associated with leaf hair development. Upstream and downstream of 120 kb to these loci (240 kb) was considered as the interval for candidate genes. Among those loci, qHL6-2 included the gene LOC_Os06g44750, which was recently reported involved in the formation of rice leaf hair. Real time RT-PCR demonstrated that transcript amounts of the gene in hairy leaf varieties were significantly higher than those with less leaf hair. Overexpression and CRISPR/Cas9 analysis of LOC_Os06g44750 confirmed its biological function in leaf hair development. Haplotype analysis on the coding region of HL6-2 in 149 rice cultivars indicated that two haplotypes were dominant among at least 7 haplotypes. In addition, the number of root hairs was found decreased in CRISPR/Cas9 plants. Our study not only verifies that combined with gene annotation, GWAS can be utilized to rapidly identify candidate genes related to leaf hair, but also provides genetic basis for dissecting molecular mechanism underlie leaf hair and root hair formation.