BackgroundMLH1epimutation is characterised by constitutional monoallelicMLH1promoter hypermethylation, which can cause colorectal cancer (CRC). Tumour molecular profiles ofMLH1epimutation CRCs were used to classify germlineMLH1promoter variants of uncertain significance andMLH1methylated early-onset CRCs (EOCRCs). Genome-wide DNA methylation and somatic mutational profiles of tumours from two germlineMLH1: c.-11C > T and oneMLH1: c.-[28A > G; 7C > T] carriers and threeMLH1methylated EOCRCs (< 45 years) were compared with 38 reference CRCs. Methylation-sensitive droplet digital PCR (ddPCR) was used to detect mosaicMLH1methylation in blood, normal mucosa and buccal DNA.ResultsGenome-wide methylation-basedConsensus Clusteringidentified four clusters where the tumour methylation profiles of germlineMLH1: c.-11C > T carriers andMLH1methylated EOCRCs clustered with the constitutionalMLH1epimutation CRCs but not with the sporadicMLH1methylated CRCs. Furthermore, monoallelicMLH1methylation andAPCpromoter hypermethylation in tumour were observed in bothMLH1epimutation and germlineMLH1: c.-11C > T carriers andMLH1methylated EOCRCs. Mosaic constitutionalMLH1methylation inMLH1: c.-11C > T carriers and 1 of 3MLH1methylated EOCRCs was identified by methylation-sensitive ddPCR.ConclusionsMosaicMLH1epimutation underlies the CRC aetiology inMLH1: c.-11C > T germline carriers and a subset ofMLH1methylated EOCRCs. Tumour profiling and ultra-sensitive ddPCR methylation testing can be used to identify mosaicMLH1epimutation carriers.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13148-023-01511-y.