The susceptibility to glyphosate and genetic diversity based on intersimple sequence repeat markers were characterized for 17 tropical sprangletop populations collected from two separate regions mainly in Persian lime groves in Veracruz, Mexico. The whole-plant dose response together with shikimic acid assays indicated different levels of glyphosate resistance in those populations. Genetic diversity values (h) estimated using POPGENE ranged from 0.119 to 0.198 and 0.117 to 0.214 within susceptible and resistant populations, respectively. The average genetic diversity (H
S) within the susceptible populations was 0.157, and the total genetic diversity (H
T) was 0.218. The H
S of the resistant populations was 0.144, and the H
T was 0.186. The analysis of molecular variance based on the response to glyphosate indicated that most of the genetic variation was found within groups of susceptible and resistant populations (90% of the genetic variation), whereas 10% or less was among groups. The high level of genetic diversity between glyphosate-resistant tropical sprangletop populations from distant and adjacent locations is likely due to both short- and long-distance seed dispersal and independent evolutionary events in tropical sprangletop populations among Persian lime groves in Veracruz.