Modern breeding programs have reduced genetic variability and might have caused a reduction in plant colonization by arbuscular mycorrhizal fungi (AM). In our previous studies, mycorrhizal colonization was affected in improved soybean genotypes, mainly arbuscule formation. Despite substantial knowledge of the symbiosis-related changes of the transcriptome and proteome, only sparse clues regarding metabolite alterations are available. Here, we evaluated metabolite changes between improved (I-1) and unimproved (UI-4) soybean genotypes and also compare their metabolic responses after AM root colonization. Soybean genotypes inoculated or not with AM were grown in a chamber under controlled light and temperature conditions. At 20 days after inoculation, we evaluated soluble metabolites of each genotype and treatment measured by GC-MS. In this analysis, when comparing non-AM roots between genotypes, I-1 had a lower amount of 31 and higher amount of only 4 metabolites than the UI-4 genotype. When comparing AM roots, I-1 had a lower amount of 36 and higher amount of 4 metabolites than UI-4 (different to those found altered in non-AM treated plants). Lastly, comparing the AM vs non-AM treatments, I-1 had increased levels of three and reduced levels of 24 metabolites, while UI-4 only had levels of 12 metabolites reduced by the effect of mycorrhizas. We found the major changes in sugars, polyols, amino acids, and carboxylic acids. In a targeted analysis, we found lower levels of isoflavonoids and alpha-tocopherol and higher levels of malondialdehyde in the I-1 genotype that can affect soybean-AM symbiosis. Our studies have the potential to support improving soybean with a greater capacity to be colonized and responsive to AM interaction.