Fall armyworm, Spodoptera frugiperda (J.E. Smith), is an important pest that damages agriculture worldwide. Organophosphorus insecticides are used for its control; however, intensive and inadequate use of insecticides has selected for resistance. The ace-1 gene confers cross resistance to organophosphorus and carbamate insecticides in cholinergic synapses. SNPs associated with resistance to organophosphorus insecticide were searched using resequencing of the ace-1 gene in populations of S. frugiperda from eight municipalities at Zacatecas, Mexico: Calera, Fresnillo, General R. Murguía, Loreto, Ojocaliente, Tepechitlán, Tlaltenango, and Villanueva. The ace-1 gene was amplified by PCR and bidirectionally sequenced in third and fifth larval instars. Results indicated the presence of the g-301 mutation in the population at Loreto, and the g-565 mutation in populations at Calera, Ojocaliente, Tepechitlán, and Villanueva (50% of the populations) with 25% homozygous and 25% heterozygous. The g-380 mutation was not observed at any town. New variations in positions g-396, g-498, and g-768 also were found. Using association analysis with a dendrogram, four groups of populations were two homozygous resistant and two homozygous susceptible. The results suggested an extensive analysis of resistance of populations in the State of Zacatecas and in Mexico to establish better strategies for controlling the pest.