Root hairs play an important role in absorbing water and nutrients in crop plants. Here we optimized high-throughput root hair length (RHL) and root hair density (RHD) phenotyping in wheat using a portable Dinolite™ microscope. A collection of 24 century wide spring wheat cultivars released between 1911 and 2016 were phenotyped for RHL and RHD. The results revealed significant variations for both traits with five and six-fold variation for RHL and RHD, respectively. RHL ranged from 1.01 mm to 1.77 mm with an average of 1.39 mm, and RHD ranged from 17.08 mm−2 to 20.8 mm−2 with an average of 19.6 mm−2. Agronomic and physiological traits collected from five different environments and their best linear unbiased predictions (BLUPs) were correlated with RHL and RHD, and results revealed that relative-water contents (RWC), biomass and grain per spike (GpS) were positively correlated with RHL in both water-limited and well-watered conditions. While RHD was negatively correlated with grain yield (GY) in four environments and their BLUPs. Both RHL and RHD had positive correlation indicating the possibility of simultaneous selection of both phenotypes during wheat breeding. The expression pattern of TaRSL4 gene involved in regulation of root hair length was determined in all 24 wheat cultivars based on RNA-seq data, which indicated the differentially higher expression of the A- and D- homeologues of the gene in roots, while B-homeologue was consistently expressed in both leaf and roots. The results were validated by qRT-PCR and the expression of TaRSL4 was consistently high in rainfed cultivars such as Chakwal-50, Rawal-87, and Margallah-99. Overall, the new phenotyping method for RHL and RHD along with correlations with morphological and physiological traits in spring wheat cultivars improved our understanding for selection of these phenotypes in wheat breeding.