Polymorphic microsatellite sequences were mined from genome sequences of two Lippia origanoides wild specimens from disjunct locations. Twenty-three primer sequences flanking the candidate markers were designed, and their usefulness for population genetic analysis was assessed in plants sampled from four municipalities in Colombia. A final set of 10 primer pairs was useful for diversity analyses, which indicated the existence of three genetic lineages in Colombia. Cross-amplification in five related Lippia species was validated. The findings supported the efficiency of in silico approaches in providing novel genetic resources for research in Lippia species.