The aim of this study was to assess the genetic diversity of four autochthonous cattle breeds of Tuscany and their relationships in comparison with Italian Friesian and Italian Brown, using amplified fragment length polymorphism markers. A total of 212 individuals were genotyped with three primer combinations generating 102 polymorphic markers. Average expected heterozygosity ranged from 0.23 in Mucca Pisana to 0.26 in Chianina, Italian Friesian, Italian Brown and Maremmana. The differences resulted not significant (Kruskall-Wallis test, p = 0.416). Gst-B index revealed that 86% of the total genetic variance is retained within population and only 14% is accounted by the between populations component. Multivariate analysis at individual and population level indicated that: (i) Calvana and Chianina are quite separate from the other breeds as an effect of the bottleneck experienced or as a signature of different origin; (ii) Podolian, Maremmana and Italian Brown clustered with the double purpose Mucca Pisana, revealing their contribution to its admixed genetic make up; (iii) Italian Friesian behaved always as out group. The 'analysis of molecular variance' recovered a significant subdivision clustering the six populations into three groups: Italian Friesian and Italian Brown versus Maremmana and Mucca Pisana versus Chianina and Calvana (6% of the total variance).