To characterize the spectrum of mosaic RB1 pathogenic alleles and map the distribution of mutant cells in available tissues from mosaic patients. Next-generation sequencing was performed on blood samples from 263 retinoblastoma families to identify mosaic RB1 variant alleles. A variety of available tissues were sampled to determine tissue distribution and fraction of mutant cells in five mosaic patients who consented to participate in mosaic pathogenic allele research. Twelve identified mosaic RB1 variants were all "null" pathogenic alleles and displayed reduced expressivity. The use of next-generation deep sequencing increased the sensitivity of mosaicism detection to 0.03% in the case of tissue DNA. In the five mosaic participants, we observed coherent but uneven, bilateral asymmetrical distribution of mutant cells across various tissues. They all carried early-embryonic mosaic pathogenic alleles and had significantly higher variant fractions in blood than in other tissues. Variant fractions of ipsilateral tissue samples were not concordant higher or lower compared with the contralateral side. Only ipsilateral conjunctival and oral epithelial cells showed concordance in mosaicism levels. No associations were observed between the laterality of affected eyes and variant fractions of any tissue type. NGS allows the detection of low-level mosaicism. Mosaic RB1 pathogenic alleles are prone to occur at very early stages of human embryonic development. With respect to genetic counseling, risk prediction should take into account unrecognized mosaicism. The underlying tissue distribution patterns of mosaic RB1 variant alleles remain to be determined.