We tested the use of biallelic Amplified fragment length polymorphism (AFLP) polymorphisms for the estimation of relative genetic distances of cattle individuals within or across breeds. An allele permutation procedure was developed to estimate the stochastic variation of the genetic distance that is inherent to a given dataset. In a panel of 47 Holstein-Friesian cattle analysed with 248 polymorphic markers, the average genetic distance of bulls selected for breeding was slightly lower than the distance of the cows. The observed standard deviation (SD) of the distance indicated genetic subdivision, which for the bulls was explained by variation in the additive relationship derived from herdbook data. Animals from three different breeds, the highly selected Holstein-Friesian, the Italian Brown and the historic Maremmana, were compared on the basis of 106 polymorphic markers. No breed-specific fragments were observed. The mean pair-wise genetic distance within breeds was 85% of the value across breeds, but principal coordinates analysis clustered the animals according to their breed of origin. Calculation of distances between the breeds indicated a relatively divergent position of the Maremmana, relative to the two other breeds. However, biallelic markers indicate that the process of breed formation had only a limited effect on the diversity at marker loci.