It has recently become possible to directly estimate the germ-line de novo mutation (DNM) rate by sequencing the whole genome of father-mother-offspring trios, and this has been conducted in human, chimpanzee, birds and fish. In these studies DNMs are defined as variants that are heterozygous in the offspring while being absent in both parents. They are assumed to have occurred in the germ-line of a parent and to have been transmitted to the offspring via the sperm or oocyte. This definition assumes that detectable mosaicism in the individual in which the mutation occurred is negligible. However, instances of mosaicism are well-documented in humans and other organisms, including ruminants. We herein take advantage of the unique pedigree structure of cattle to show that mosaicism associated with DNMs is a common occurrence, and that this should be taken into account to accurately estimate the mutation rate in this and possibly other species. It suggests that early cleavage cell divisions are particularly mutation-prone, and that the recurrence risk of DNM-dependent disorders in sibs may be higher than generally assumed.