Trimerotropis pallidipennis is an American grasshopper whose South American populations are polymorphic for pericentric inversions. Colonization of southern latitudes was by North American grasshoppers with basic chromosome arrangements, presumably along the Andean dry lands of South America. In Argentina, the frequencies of some of the rearrangements are correlated with geographical and climatic variables, following similar patterns among different ecological gradients, and are probably maintained by geographically variable coefficients of selection. Restriction site variation of mitochondrial DNA is used as a tool for determining the species history in relation to the formation of clines. Populations located along an altitudinal gradient, and others outside the cline, are analysed through phylogeographical studies. There is no strong geographical orientation in the unrooted tree connecting all 17 mitochondrial DNA haplotypes found. Many of them are present in most of the populations analysed, indicating high gene flow. The fact that there is no obvious differentiation in haplotype distribution between both extremes of the cline nor between chromosomally differentiated populations shows that the cline is not the result of a hybrid zone and reinforces the selection hypothesis. The estimation of the overall nucleotide divergence between the hypothetical ancestral haplotype and the other molecules shows that T. pallidipennis haplotypes started diverging from each other about 3 Myr ago. This result is in agreement with the time when the Isthmus of Panamá rose (2–3 Myr ago), probably favouring the migration of many species between both hemispheres.