The root system architecture (RSA) determines the composition of the root system and plays an irreplaceable role in crop growth and development. It is of great significance to analyze the genetic mechanism of wheat root architecture and the selection of varieties with better RSA traits in breeding for elite wheat varieties. Genome-wide association study (GWAS) is an effective method to analyze the genetic mechanism of complicated quantitative traits (i.e. RSA) in wheat. In the study, GWAS for RSA-related traits(total root length, total root surface area, total root volume, average root diameter and number of root tips) were conducted in 160 wheat accessions from Henan and Shandong, which were previously genotyped by wheat 660K SNP chip. Twenty-three loci associated with RSA related traits were detected from chromosomes 1A, 2A, 2B, 3B, 4A, 5A, 5B, 5D, 6A, 6B and 7B, and each explained the phenotypic variation variable from 7.2% to 12.8%. Among them, eleven loci are found to coincide with the formerly-reported loci, and the remaining 12 loci are newly identified. Taken together, this study is of significance to uncover the genetic structure of the root architecture with a potential in future breeding of high-yield and stress-resistant wheat cultivars.