Native polyacrylamide gel electrophoresis was used to identify polymorphisms in α- and β-esterases loci and electrophoresis in starch gel to identify polymorphism in malate dehydrogenase (MDH; EC 220.127.116.11) and acid phosphatase (ACP; EC 18.104.22.168) isozymes loci in leaf tissues from samples of horseweed and hairy fleabane populations to determine genetic diversity and population structure. Similar or differential genetic divergence between the two species may guide specific use of herbicides. For samples of plants with high genetic similarity it is possible to adopt similar mechanisms and processes for their control. The proportion of polymorphic loci was 57.14, 50.0, and 53.6%, in samples of horseweed and hairy fleabane, for EST, MDH, and ACP isozymes, respectively. A comparison of the diversity parameters in the two species showed that the number of alleles is similar in the horseweed and hairy fleabane plants. The estimated heterozygosity in horseweed and hairy fleabane was also very close. A relatively low level of population differentiation was detected between horseweed and hairy fleabane (FST = 0.0199), which suggests a substantial genetic exchange among the two species. Accordingly, estimate of gene flow was high (Nm = 12.3172) for the alleles of the loci Est, Mdh, and Acp. The Nei's identity (I) values also was high (I = 0.9561) indicating very high similarity between the two Conyza species. AMOVA showed higher genetic variation within (95%) than among (5%) the two samples. The low genetic structure and high value of genetic identity was an important indication that alleles are exchanged between horseweed and hairy fleabane populations, and provides additional evidence of occurrence of outcrossing between populations or dispersion of samples of one for other site.