The genetic diversity and population structure of Lippia origanoides, a species of the Verbenaceae family that shows promise as a crop plant, was investigated along an altitudinal gradient in the basin of the Chicamocha River in northeastern Colombia. The economic importance of the species, quality of its essential oils, and the fact that it is restricted to some few semiarid areas in northern South America may put the species at risk in a scenario of uncontrolled harvest of natural populations. Lippia origanoides was sampled along an altitudinal gradient from 365 to 2595 m.a.s.l. throughout Chicamocha River Canyon, a semiarid area in northeastern Colombia. Genetic diversity was assessed by means of AFLP markers. The number of AFLP loci (355) and the number of individuals sampled (173) were sufficient to reliably identify four populations at contrasting altitudes (FST = 0.18, P-value < 0.0000), two populations in the lower basin, one population in the medium basin, and one population in the upper basin, with a low level of admixture between them. In average, genetic diversity within populations was relatively high (Ht = 0.32; I = 0.48); nevertheless, diversity was significantly reduced at higher altitude, a pattern that may be consistent with a scenario of range expansion toward higher elevations in an environment with more extreme conditions. The differences in altitude among the basins in the Chicamocha River seem to be relevant in determining the genetic structure of this species.