Root system morphology influences the absorption efficiency of soil water and nutrient, and therefore has a direct impact on abiotic stress tolerance in crop plants, but the genetic basis of root characters affecting wheat resilience to abiotic stress remain to be elucidated. In the present study, we investigated phenotypic differences in root traits and shoot traits of 323 wheat accessions and 150 doubled haploid lines, and performed a genome-wide association study (GWAS) with 395,681 single nucleotide polymorphisms. Relationships among multiple root traits and stress tolerances were identified, among which, the correlation coefficient between root depth and drought tolerance was relatively high. Ten loci associated with root depth at booting and/or mid-grain fill stages were detected by mixed linear model (MLM), among which three hub loci were simultaneously associated with canopy temperature (CT) and yield per plant (YPP). The YPP was significantly higher in genotypes which combined favorable marker alleles (FMAs) for root depth and PH, suggesting that deep-root and shorter plant height are suitable traits for pyramiding target alleles by molecular marker assisted breeding. In addition, one peak GWAS signal on chromosome 2B showed that a 125 bp miniature inverted-repeat transposable element (MITE) in the promoter of TaVSR1-B, encoding a vacuolar sorting receptor protein, contributes significantly to root depth at the booting stage, and a candidate gene (TaARN) regulating adventitious root number were identified by combined GWAS and biparental QTL mapping. These results uncovered promising candidate genomic regions and functional genes for the dissection of molecular mechanisms regulating root traits in wheat, enhanced understanding of correlation between root and abiotic stress tolerance, and will facilitate intensive study on root morphology and breeding through molecular design.