Objective To identify the pathogenic mutation in a three generation Chinese family with low penetrance retinoblastoma (RB). Methods 8 from 9 family members received complete ophthalmic examinations. DNA was extracted from 6 family members. Using exon combined target region capture sequencing chip to screen the candidate disease-causing mutations. Sanger sequencing were used to confirm the disease-causing mutation. Results Among 9 family members, the proband (Ⅲ2) was bilateral RB, Ⅲ1 was unilateral RB, Ⅲ3 was dead for bilateral RB. Normal fundus were observed in the left eye of Ⅲ1 and the eyes of other family members except the proband. Sequence analysis of RB1 gene revealed a missense mutation c. 1981C>T (p.Arg661Trp) in the proband and two carriers (Ⅱ2, Ⅱ3), but not in the two normal subjects (Ⅱ1, Ⅱ4). We suspect that the RB penetrance in the family was 50%. Conclusions There is a missense mutation c. 1981C>T in a Chinese family with low penetrance RB. The RB penetrance is 50%. Key words: Retinoblastoma; Genes, neoplasm; Penetrance; High-throughput nucleotide sequencing