The aim of this study was to evaluate the methane (CH4) emission of Santa Inês sheep fed cottonseed by-products, verifying if the gossypol content of these feedstuffs affects CH4 emission. Twelve late-lactating Santa Inês sheep (44.8 ± 7.5 kg body weight (BW)) were allocated in metabolic cages for an experimental period of 19 days, 14 days for adaptation and 5 days for measuring CH4 emission and dry matter intake (DMI). The animals were divided into four treatments, established in accordance with the cottonseed by-product used in concentrate formulation: Control (CON - no cottonseed by-product), Whole cottonseed (WCS), Cottonseed cake (CSC), and Cottonseed meal (CSM). The free gossypol level of the concentrates were 0, 1,276, 350, and 190 ppm for CON, WCS, CSC, and CSM, respectively. Also, the animals received Cynodon dactylon cv. Coast Cross hay, water, and mineral salt ad libitum. The ether extract content of the diets was balanced between treatments by including soybean oil in concentrates. The technique used to measure the CH4 emission was the sulfur hexafluoride (SF6) tracer technique, and the gas samples collected were quantified by analysis in gas chromatography system. The CH4 emission was evaluated considering the daily emission (g CH4/day); DMI (g CH4/kg DMI); and BW (g CH4/kg BW). No statistical difference was found (P > 0.05) between treatments for DMI and CH4 parameters. In the regression analysis, no significant relation (P > 0.05) between gossypol content and CH4 emission was observed. These results suggest that gossypol does not affect rumen methanogenesis.