The technique for obtaining mitotic chromosomes proposed by Foresti et al. (1981) when applied in freshwater stingrays didn’t result in good quality chromosome preparations. Only a few studies have used this technique to obtain good quality chromosomes in this fi sh group (Valentim et al. 2006; Cruz et al. 2011). Therefore, it was necessary to adapt the technique to freshwater stingrays. Classical in vivo methods basically consist of mitotic stimulation followed by colchicine injection, according to the technique described in Chapter 2. In this chapter we present modifi cations of this technique to obtain a better quality of chromosomes in stingrays. Typically, the kidney tissue is used in this technique, but in stingrays the tissue that showed the best quality and quantity of metaphases was the spleen. Other important modifi cations are the time of colchicine and hypotonic treatments.