The Lebranche mullet (Mugil liza) is an economically important estuarine species found along the coast of Brazil. The southern population of this species ranges from the coast of the state of São Paulo (23°S) to the coast of Argentina (36°S). It migrates annually among estuaries in Argentina, Uruguay, and southern Brazil to reproduce in their marine spawning grounds (~26°S). We evaluated whether variations in the otolith chemical composition for fish acquired at nursery grounds can be used to make distinctions among 4 nursery areas of the southern population. Analysis of otolith microchemistry included concentrations of 9 elements: lithium (Li), magnesium (Mg), copper, zinc, strontium (Sr), cadmium, barium, lanthanum, and lead. When using random forest classification algorithms, the maximum accuracy of 96% in assignment of nursery habitat for Lebranche mullet between 2 groups (fish caught at nurseries in Brazil and fish migrating from waters of Uruguay and Argentina) was achieved with the combination of Li, Mg, and Sr. Our results indicate that the elemental composition of otoliths can be an important tool for establishing connectivity between nursery areas used by Lebranche mullet. We discuss the implications of this result for the structure of the population and the management of the mullet fishery in southern Brazil relative to the limitations of the methods we employed.