<p>Genetically modified (GM) soybean (<i>Glycine max</i>) expressing a human thioredoxin (<i>trx</i>) gene under the control of a seed-specific promoter has been developed for cosmetic applications, but its ecological effects remain poorly understood. We examined the rhizosphere microbiomes of GM soybean, wild soybean (<i>Glycine soja</i>), and F₃ interspecific hybrids segregating for the transgene under low-input field conditions. Rhizosphere soil samples were collected at the vegetative and flowering stages, and microbial communities were analyzed via high-throughput sequencing of 16S rRNA and ITS regions. This study represents the first application of amplicon sequence variant (ASV)-level niche breadth analysis to evaluate the rhizosphere effects of a <i>trx</i>-expressing GM soybean. ASV-level analysis and ecological niche breadth classification revealed that genotype-specific microbial shifts were not apparent at relatively high taxonomic levels. Notably, several bacterial ASVs from the class <i>Bacilli</i> were more abundant in GM and homozygous plants at flowering. A fungal ASV from <i>Tausonia</i> also showed increased abundance in the GM lines. These findings highlight that genotype-driven microbial shifts can occur in a stage-specific manner and underscore the importance of fine-resolution microbial analyses in environmental risk assessment.</p>