Northwest China is closely adjacent to Central Asia, an intermediate region of the Eurasian continent. Moreover, the Silk Road through the northwest of China once had a vital role in the east-west intercommunications. Nevertheless, little has been known about the genetic makeup of populations in this region. We collected 503 male samples from 14 ethnic groups in the northwest of China, and surveyed 29 Y-chromosomal biallelic markers and 8 short tandem repeats (STRs) loci to reconstruct the paternal architecture. Our results illustrated obvious genetic difference among these ethnic groups, and in general their genetic background is more similar with Central Asians than with East Asians. The ancestors of present northwestern populations were the admixture of early East Asians peopling northwestward and later Central Asians immigrating eastward. This population mixture was dated to occur within the past 10 000 years. The J2-M172 lineages likely entered China during the eastward migration of Central Asians. The influence from West Eurasia through gene flows on the extant ethnic groups in Northwest China was relatively weak.