Persistent high-risk human papillomavirus (HR-HPV) infections cause cervical cancer and a fraction of head and neck cancer. To investigate whether HR-HPV infection might be also involved in the development of gastric cancer (GC), we developed a platform utilizing a rolling circle amplification (RCA)-based nested L1 polymerase chain reaction with Sanger sequencing to genotype the HPV DNA in cancer tissues of 361 GC and 89 oropharyngeal squamous cell carcinomas (OPSCC). HPV transcriptional activity was determined by E6/E7 mRNA expression and a 3' rapid amplification of cDNA ends was performed to identify HPV integration and expression of virus-host fusion transcripts. Ten of 361 GC, 2 of 89 OPSCC, and 1 of 22 normal adjacent tissues were HPV L1 DNA-positive. Five of the 10 HPV-positive GC were genotyped as HPV16 by sequencing and 1 of 2 GC with RCA/nested HPV16 E6/E7 DNA detection exhibited HPV16 E6/E7 mRNA. Two OPSCC displayed HPV16 L1 DNA and E6/E7 mRNA, of which 1 OPSCC tissue showed virus-host RNA fusion transcripts from an intron region of KIAA0825 gene. Together, our data reveal viral oncogene expression and/or integration in GC and OPSCC and a possible etiology role of HPV infections in gastric carcinogenesis.