The use of cotton co-products in animal feed is restricted by the presence of gossypol, which is a toxic and highly reactive molecule of complex minerals. In mammals, part of the offspring phenotype is influenced by dam nutrition. The aim of this study was to investigate the effect of ewe diet, with and without cottonseed (gossypol), on the testicular development of lambs from birth to weaning through the assessment of lamb live weight (LW), macro and histological morphology of testes, and mass fraction of chemical elements by neutron activation analysis (NAA) and microprobe X-ray fluorescence spectroscopy, as well as the multielement distribution map in the testes. Eighteen lambs were used with an average LW at birth of 4 ± 1.0 kg. All lambs were offspring of Santa Inês ewes, fed on ration either with or without cottonseed during mating, gestation, and lactation, thus forming two treatments: control group (C) without cottonseed and treatment group with cottonseed (G). The animals were weighed from birth to 60 days of age, at fortnightly intervals. At 60 days of age, the lambs were orchidectomized to collect their testes for macroscopy, histological, neutron activation, and X-ray fluorescence analysis. Besides dry matter (DM), protein, ether extract (EE), calcium (Ca), and potassium (K) were higher in the ewe milk from the C group compared with the G group ( P  < 0.05). Lambs from the C group showed higher LW from 45 days onwards, as well as higher average daily gain when compared with the G group ( P  < 0.05). They also presented higher testicular weight, volume, length, width, as well as tubule and lumen diameters compared with lambs from the G treatment ( P  < 0.05). Multielementary NAA revealed higher mass fractions of rubidium (Rb), selenium (Se), and cesium (Cs) in the testes of lambs from the C group when compared with the G group, while G showed higher zinc (Zn) content ( P  < 0.05). No differences between treatments were found for element levels and distribution using X-ray fluorescence microanalysis ( P  > 0.05). In conclusion, the maternal cottonseed diet compromised performance and testes development of the lambs and reduce the content of Se in the testes.