The trophic ecology of the Pelagic Thresher shark (Alopias pelagicus) was evaluated based on chemical ecology using stable isotope ratios of carbon (δ13C) and nitrogen (δ15N) in the vertebrae and muscles. Individuals were caught between August 2013 and October 2019 on both the coasts of Baja California Sur, Mexico. In Bahía Tortugas, the mean vertebrae (n = 35) values were 12.72 ± 1.06‰ (δ15N) and −14.79 ± 0.61‰ (δ13C), while in muscles (n = 32) these values were 16.63 ± 0.76‰ (δ15N) and −17.18 ± 0.39‰ (δ13C). In Santa Rosalía, the mean vertebrae (n = 125) isotopic values were 14.4 ± 1.59‰ (δ15N) and −14.18 ± 0.51‰ (δ13C), while in muscles (n = 43), these values were 18.08 ± 0.96‰ (δ15N) and −16.43 ± 0.34‰ (δ13C). These results show higher δ15N values in Santa Rosalía as an effect of baseline isotopic differences between the two regions, whereas the δ13C values were lower in Bahía Tortugas, suggesting offshore ecological behavior (p < 0.05). In Santa Rosalía, there were significant differences by sex for δ15N in muscle, whereas the δ13C showed ontogenetic shifts, indicating that neonates feed in coastal areas more commonly than juveniles or adults (p < 0.05). Neither sex nor ontogenetic differences were observed in Bahía Tortugas (p > 0.05), suggesting a high overlap between their isotopic niches. Therefore, Alopias pelagicus uses the same ecological niche throughout its life, and there is consistency between sexes. The mean trophic position for both tissues and regions was 4.5, which corresponds to a tertiary predator, without any differences between stages or sex. Due to their higher energetic needs, juveniles and females showed the greatest isotopic niche amplitude; thus, their ecological niche is the widest.