Cytogenetic evaluation is an important step in the diagnosis of infertile or sterile animals. Moreover, the analysis of sex chromosomes is crucial for a proper classification of disorders of sex development DSD . For many years, chromosome studies mainly addressed the livestock species, while recently, increasing interest in such analysis in companion animals is observed. New molecular and cytogenetic tools and techniques have given opportunities for a precise identification of chromosome mutations. Among them, fluorescence in situ hybridization, besides chromosome banding, has become a gold standard. In this chapter, recent advances in the cytogenetic diagnosis of cattle, pigs, horses, dogs and cats are presented.