Fecal sampling is a widely used, non-invasive method for assessing gut microbiomes across various organisms. However, its suitability for studying the gut microbiome of honey bee ( Apis mellifera ) queens has not been tested. In this study, we evaluated whether fecal microbiomes accurately reflect gut microbiomes in honey bee queens, offering a potential non-destructive approach for microbiome research. We successfully obtained fecal and gut samples from 21 out of 26 queens. Bacterial communities were analyzed using 16S rRNA amplicon sequencing and qPCR. Our results indicate that queen fecal microbiomes closely resemble gut microbiomes, with no significant differences in alpha diversity and only minor differences in specific bacterial taxa. Beta diversity analyses revealed that within-pair microbiomes (i.e., gut vs. feces from the same queen) were significantly more similar than between-pair comparisons. Additionally, qPCR analyses revealed a strong positive correlation between bacterial abundances in fecal and gut samples, further supporting the use of feces as a proxy for gut microbiome composition. While promising, fecal collection from queens can sometimes be challenging. In our study, we were unable to collect feces from five queens, and these individuals lacked stored fecal material upon inspection of dissected guts. Nonetheless, our findings suggest that fecal sampling can be a useful, non-invasive method for studying honey bee queen microbiomes, enabling longitudinal assessments without compromising colony stability.