Multiple biotypes of soybean aphid, Aphis glycines, occur in North America adapted for survival (virulence) on soybean, Glycine max, with one or more different resistance to A. glycines (Rag) traits. The degree of genome-wide variance between biotypes and the basis of virulence remains unknown, but the latter is hypothesized to involve secreted effector proteins. Between 167,249 and 217,750 single nucleotide polymorphisms (SNPs) were predicted from whole genome re-sequencing of A. glycines avirulent biotype 1 (B1) and virulent B2, B3 and B4 colony-derived iso-female lines when compared to the draft B1 genome assembly, Ag_bt1_v6.0. Differences in nucleotide diversity indices (π) estimated within 1000 bp sliding windows demonstrated that 226 of 353 (64.0%) regions most differentiated between B1 and ≥ 2 virulent biotypes, representing < 0.1% of the 308 Mb assembled genome size, are located on 15 unordered scaffolds. Furthermore, these 226 intervals were coincident and show a significant association with 326 of 508 SNPs with significant locus-by-locus FST estimates between biotype populations (r = 0.6271; F1,70 = 45.36, P < 0.001) and genes showing evidence of directions selection (πN/πS > 2.0; r = 0.6233; F1,70 = 50.20, P < 0.001). A putative secreted effector glycoprotein is encoded in proximity to genome intervals of low estimated π (putative selective sweep) within avirulent B1 compared to all three virulent biotypes. Additionally, SNPs are clustered in or in proximity to genes putatively involved in intracellular protein cargo transport and the regulation of secretion. Results of this study indicate that factors on a small number of scaffolds of the A. glycines genome may contribute to variance in virulence towards Rag traits in G. max.