The root tissues play important roles in water and nutrient acquisition, environmental adaptation, and plant development. In this study, a diversity panel of 388 wheat accessions was collected to investigate nine root system architecture (RSA) traits at the three-leaf stage under two growing environments: outdoor pot culture (OPC) and indoor pot culture (IPC). Phenotypic analysis revealed that root development was faster under OPC than that under IPC and a significant correlation was observed between the nine RSA traits. The 660K single-nucleotide polymorphism (SNP) chip was used for a genome-wide association study (GWAS). Significant SNPs with a threshold of −log10 (p-value) ≥ 4 were considered. Thus, 36 quantitative trait loci (QTLs), including 13 QTL clusters that were associated with more than one trait, were detected, and 31 QTLs were first identified. The QTL clusters on chromosomes 3D and 5B were associated with four and five RSA traits, respectively. Two candidate genes, TraesCS2A01G516200 and TraesCS7B01G036900, were found to be associated with more than one RSA trait using haplotype analysis, and preferentially expressed in the root tissues. These favourable alleles for RSA traits identified in this study may be useful to optimise the root system in wheat.