Human ageing has been predicted to be caused by the accumulation of molecular damage in cells and tissues. Somatic mitochondrial DNA (mtDNA) mutations have been documented in a number of ageing tissues and have been shown to be associated with cellular mitochondrial dysfunction. It is unknown whether there are selective constraints, which have been shown to occur in the germline, on the occurrence and expansion of these mtDNA mutations within individual somatic cells. Here we compared the pattern and spectrum of mutations observed in ageing human colon to those observed in the general population (germline variants) and those associated with primary mtDNA disease. The pathogenicity of the protein encoding mutations was predicted using a computational programme, MutPred, and the scores obtained for the three groups compared. We show that the mutations associated with ageing are randomly distributed throughout the genome, are more frequently non-synonymous or frameshift mutations than the general population, and are significantly more pathogenic than population variants. Mutations associated with primary mtDNA disease were significantly more pathogenic than ageing or population mutations. These data provide little evidence for any selective constraints on the occurrence and expansion of mtDNA mutations in somatic cells of the human colon during human ageing in contrast to germline mutations seen in the general population.