We quantified the δ13C and δ15N values in the lower beaks of Humboldt squid, Dosidicus gigas, collected from international waters off Costa Rica, Ecuador, Peru and Chile by Chinese squid jigging vessels during 2009, 2010 and 2013. There was a significant difference in the isotopic values among regions with the lowest value off Ecuador and the highest off Chile, which were interpreted as a function of trophic effects as well as baseline values. However, constant trophic level of D. gigas across its geographic range showed that spatial variation in the baseline of primary production is the main driver responsible for the observed geographic isotope variability. Inter-regional difference and intra-regional convergence of isotope values indicated squid off Costa Rica, Ecuador and Chile belong to different geographically segregated populations, which were previously proved by integrated population identifying method. In contrast, the higher variations in δ13C and δ15N values in a given size group suggest the squid off Peru move and forage in different places. Moreover, potential population exchange could be responsible for the overlap of the isotope values between the squid off Peru and off Chile. On the whole, the spatial difference in isotopic values of Humboldt squid beaks improves our understanding of potential geographic population connectivity and movement.