The objective of this work was to evaluate the influence of slaughter weight on the expression of calpastatin, m-calpain and the RyR3 gene, and on the chem. composition, morphometric measurements, fillet yield and sensorial characteristics of Nile tilapia fillets. In the experiment, 90 Nile tilapia were divided into three exptl. treatments regarding slaughter weight (n = 30): in treatment 1, tilapia aged 140 days were slaughtered with an average body weight of 665 ± 85 g; in treatment 2, the animals were slaughtered at 182 days and weighed 1000 ± 177 g; and in treatment 3, they were slaughtered at 238 days and weighed 1325 ± 167 g. There was no significant difference (P > .05) between the treatments for the chem. composition and fillet yield. Fillets of tilapia slaughtered with a weight of 665 g presented higher expression of m-calpain and lower expression of calpastatin gene, lower pH values of the thawed fillet, lower drip and thawing loss, and lower shear force than animals slaughtered with the highest evaluated weight Tilapias slaughtered at 665 g also presented higher flavor and general acceptance. These results show that slaughter weight may influence important aspects of the quality of Nile tilapia fillets and that the slaughter of Nile tilapia with a body weight of 665 g allows fillets that serve the consumer market to be obtained.