Retinoblastoma (RB) is a rare childhood malignant disorder caused by the biallelic inactivation of the RB1 gene. Early diagnosis and identification of carriers of heritable mutations in RB1 can improve disease outcome and management. In this study, we present the spectrum of mutations in the RB1 gene in Vietnamese patients with RB.Tumor RNA from 50 probands with RB, including 12 bilateral and 38 unilateral cases, was extracted. cDNA, after reverse transcription, was sequenced to identify the RNA mutation of the RB1 gene. At the genomic DNA level, mutational analysis of all RB1 exons, exon-intron boundaries, and the promoter region was conducted using PCR and direct sequencing. Multiplex ligation-dependent probe amplification (MLPA) analysis was performed for patients for whom the first two results were negative. For patients for whom either the sequencing or MLPA results were positive for a tumor mutation, patients' and their parents' blood DNA was analyzed to determine the germline mutation.Forty-one different kinds of RB1 tumor mutations were identified in 41 probands (82.0%), including 11 of 12 bilateral cases (91.7%) and 30 of 38 unilateral cases (78.9%). The majority of the detected mutations were nonsense (15 different kinds), followed by frameshift (11 kinds), and splice site mutations (nine kinds). Each splice site mutation was confirmed to create a deletion of the corresponding exon with RNA sequencing. The single promoter mutation c.-197G>A was reported previously; however, both missense mutations identified in exon 6 (c.601G>C: p.A201P) and exon 22 (c.2264T>C: p.F755S) were novel. Gross deletions were detected with MLPA in three probands. The detection rate of germline mutations in bilateral and unilateral cases with mutations were 81.8% and 30.0%, respectively. Only one father out of the 20 parents tested was positive for a germline mutation.Mutations in the RB1 gene in Vietnamese patients were heterogeneous and highly prevalent with pathogenic truncated mutations. With advancement in therapeutics, early detection of RB is important for eye salvation.