Background Recent advancement in speciation biology proposes that genetic differentiation across the whole genome (genomic differentiation, GD) may occur at the beginning of a speciation process and that GD itself may accelerate the rate of speciation. The fall armyworm (FAW, Spodoptera frugiperda) has been used as a model species to study the process of speciation between diverging host-plant strains. We showed in a previous study that GD between the host-plant strains occurred at the beginning of a speciation process based on a population genomics analysis from a population in Mississippi (USA), providing empirical support for the theoretical prediction. In a recent paper, however, panmixia was reported in FAW based on the genomic analysis of 55 individuals collected from Argentina, Brazil, Kenya, Puerto Rico, and the mainland USA. If panmixia is true, the observed differentiation in Mississippi could be at most a phenomenon specific to a geographic population, rather than a status during a speciation process. In this report, we reanalyzed the resequencing data to test the existence of population structure according to host plants using different bioinformatics pipelines. Results Principal component analysis, F ST statistics, and ancestry coefficient analysis supported genetic differentiation between strains regardless of the used bioinformatics pipelines. The strain-specific selective sweep was observed from the Z chromosome, implying the presence of strain-specific divergence selection. Z chromosome has a particularly high level of genetic differentiation between strains, while autosomes have low but significant genetic differentiation. Intriguingly, the re-sequencing dataset demonstrates the spread of Bacillus thuringiensis resistance mutations from Puerto Rico to the US mainland. Conclusions These results show that a pair of host-plant strains in FAW experience genomic differentiation at the beginning of a speciation process, including Z chromosome divergent selection and possibly hitchhiking effect on autosomal sequences.