The methylation of CpG sites in the promoter region of the P16 gene in Uyghur patients with cervical squamous cell carcinoma (CSCC) was quantitatively analyzed and its relationship with human papillomavirus 16 (HPV16) infection was explored. Cervical samples were collected from 20 Uyghur patients with CSCC and 20 Uyghur controls. Matrix-assisted laser desorption ionization-time of flight mass spectrometry was applied to detect methylation of CpG sites in the promoter region of the P16 gene; polymerase chain reaction was performed to assess HPV16 infection in the 2 groups. Among the 16 CpG sites in the P16 gene promoter region, the methylation level of the CpG1-2 and CpG 6 sites, as well as the HPV16 infection rate, was higher in the CSCC group than in the control group (P<0.05). There was no significant correlation between P16 CpG methylation and HPV16 infection in Uyghur patients with CSCC. The P16 gene CpG1-2 and CpG 6 hypermethylation and HPV16 infection, which are independent of each other, play an important role in cervical squamous cell carcinogenesis in Uyghur patients.