<h3>Purpose/Objective(s)</h3> This study investigates the association between pre-treatment positron emission tomography (PET) metrics and clinical outcomes in anal squamous cell carcinoma (ASCC) patients treated with definitive radiation therapy (RT). We hypothesize that pre-treatment PET metrics may differentially predict clinical outcomes in ASCC patients without HIV infection (HIV-) and ASCC patients living with HIV (PLWH) with detectable or undetectable viral load (VL). <h3>Materials/Methods</h3> Patients treated with definitive RT for non-metastatic ASCC between 2005 - 2021 at a single institution were retrospectively identified, and clinical and demographic data, including HIV status and pretreatment VL, were tabulated. Pre-treatment PETs were imported to MIM, and semiautomatic gradient-based segmentation tool algorithms (PET_Edge) were used to calculate PET metrics, including maximum standardized uptake value (SUVmax), metabolic tumor volume (MTV), and total lesion glycolysis (TLG). Cox proportional hazard modeling was used to investigate the association between primary tumor PET metrics and clinical outcomes, including overall survival (OS), progression free survival (PFS), and loco-regional control (LRC) <h3>Results</h3> 141 ASCC patients were identified, 49 of whom were PLWH. 12 had detectable HIV VL. The median follow-up time was 51.8 months (IQR: 0.5-191.2 months). There were 22 loco-regional failures and 24 deaths. 3- and 5- year LRC was 85.6% and 83.3%, 3- and 5- year PFS was 75.9% and 71.6%, and 3- and 5- year OS was 89.3% and 86.6% for the entire cohort. There was no significant difference in LRC, PFS, or OS between PLWH and HIV- patients (p>0.50). PLWH with detectable VL had similar LRC and PFS but trended towards worse 5-year OS than did HIV- patients (66.3% vs 89.7%, p=0.16). MTV and TLG from primary tumors were significantly associated with LRC, PFS, and OS for all patients and in sub-group analysis for HIV-, PLWH, and PLWH with undetectable VL (p<0.05). SUVmax was significantly associated with LRC, PFS, and OS for all patients, and on sub-group analysis only for HIV- patients (p<0.05). On multivariable analysis, after adjusting for stage and Charlson Comorbidity Index, MTV split at median was significantly associated with LRC (HR 3.59 (95% CI:1.21-10.6), p=0.02) and PFS (HR 3.12 (95%CI:1.48-6.56), p=0.003), and TLG split at median was significantly associated with LRC (HR 4.36 (95%CI:1.36-14.0), p=0.01) and PFS (HR 3.13 (95%CI:1.48-6.64), p=0.003). <h3>Conclusion</h3> Patients with ASCC had similar outcomes regardless of HIV status. Advanced pre-treatment PET metrics, especially MTV and TLG, may have prognostic or risk-stratification utility in HIV- patients and undetectable PLWH with ASCC, but were not as strongly associated with clinical outcomes for PLWH with detectable VL, perhaps due to small numbers of patients with detectable VL in this cohort.