<h3>Purpose/Objective(s)</h3> There is an increasing interest in the composition of tumor-associated microbiomes and their effect on treatment response and patient outcomes. This study aims to explore the differences between the tumor-associated microbiome landscapes of patients diagnosed with HPV-related anogenital cancers. We have previously shown that <i>Lactobacillus iners</i> is associated with poor treatment response among cervical cancer patients, so we studied the presence of <i>L. iners</i> among other anogenital cancers. <h3>Materials/Methods</h3> Tumor swabs were collected from patients diagnosed with cervical cancer (n=85), anal cancer (n=20), vaginal cancer (n=7), and vulvar cancer (n=3) receiving chemoradiation therapy (CRT) from 2 institutions. Samples were collected and analyzed at baseline, week 1, 3, and 5 during CRT and at 3 months post-CRT using 16Sv4 rRNA gene sequencing. Multivariate community-level differences between the tumor microbiomes was conducted using principal coordinates analysis (PCoA) plots and pairwise PERMANOVA. Linear Discriminant Effect Size (LEfSe) analysis was used to identify specific taxa abundant among the different tumor types at baseline and end of treatment. <h3>Results</h3> PERMANOVA revealed significant compositional differences between the cancer types (p= 0.001). There were significant compositional differences between all cancer types (q<0.01 for all), except between vaginal and vulvar cancers (q= 0.12). LEfSe revealed that the microbiome of each cancer was dominated by different bacteria. <i>L. iners</i> was enriched in cervical samples, Clostridia and Bacteroidaceae were enriched in anal samples, Proteobacteria was enriched in vaginal samples, and Peptostreptococcales-Tissierellales and Staphylococcales were enriched in vulvar samples at baseline. Moreover, by the end of treatment, Bifidobacteriales were enriched in cervical samples, while Proteobacteria, Corynebacteriales, and Clostridia were enriched in anal samples, and Bacteroidota was enriched in vulvar samples. Of interest, L. iners was present in two out of three non-responders with anal cancer, and one out of two non-responders with vaginal cancer. However, the proportions of Liners in these patients were less than those in patients with cervical cancer. <h3>Conclusion</h3> Our results show that the tumor microbiomes of HPV-related anogenital cancers differ significantly from each other and have distinct reactions to CRT. These findings could lay the groundwork for interventions tailored to the tumor-specific microbiome to enhance the effectiveness of CRT.