The soybean aphid (Aphis glycines Matsumura) is the most important insect pest posing a threat to soybean [Glycine max (L.) Merr.] grain production in the United States. Soybean cultivars with resistance are currently being deployed to aid in management of the pest. Temperature has been reported to influence the expression of host plant resistance against crop pests. The objective of this study was to determine if temperatures, 14, 21, and 28°C, altered resistance expression in four aphid‐resistant soybean genotypes, LD05‐16611 (with the Rag1 resistance gene), PI 200538 (Rag2), PI 567541B (rag1c, rag4), and PI 567597C (antixenosis) compared to a susceptible soybean genotype (Williams 82) when challenged with soybean aphid. A replicated no‐choice experiment was conducted and aphid populations of the three soybean aphid biotypes, 1, 2, and 3, were enumerated 14 d after inoculating the five soybean genotypes. Significant interactions were found in the analysis of the effects of temperature, soybean genotype, and aphid biotype on aphid populations. Responses between soybean genotypes and aphid biotypes were consistent with previous reports with the exception of PI 567541B, which had lower resistance against aphid biotype 1 than found in an earlier study. At 14°C, there was limited aphid population growth on all soybean genotypes, including Williams 82. At 21 and 28°C, resistance expression was stable in LD05‐16611 (Rag1), PI 567541B (rag1c, rag4), and PI 567597C (antixenosis), relative to susceptible Williams 82; however, resistance expressed in PI 200538 (Rag2) was significantly stronger at 28°C than at 21°C, as indicated by differences in aphid population size that were found. Results of this study indicated that soybean aphid virulence variability had a stronger influence on the effectiveness of soybean aphid resistance than did temperature, and that the aphid resistance tested in this study would remain stable or be stronger in temperatures ranging between 21 and 28°C.