In this study we explored the mutation types of p16CDKN2A exon 1 and the corresponding frequencies in experimental rat tongue carcinogenesis. Twenty barrier Sprague-Dawley (SD) rats were divided into the control (n = 5) and experimental group (n = 15), to which 4-nitroquinoline-1-oxide (4-NQO) in drinking water was administered. Two samples of normal, three samples of moderate/severe dysplasia and four samples of invasive squamous cell carcinoma lesions were selected following strict histopathological examination in double-blind manner. The PCR products of p16CDKN2A exon 1 amplified from these tissues were sequenced. Point mutations of p16CDKN2A exon 1 were found in all of the precancerous and cancerous lesions. Half of the mutations were detected on guanine (G). Twenty mutations, including a missense mutation of the start codon resulting in alternative reading frame of p16 CDKN2A exon 1, were also identified. These preliminary results suggested that mutation of p16CDKN2A exon 1 might be an early molecular event of rat tongue carcinogenesis induced by 4NQO and G was the mutation hotspot.