Background: We hypothesize that circulating tumor DNA (ctDNA) dynamics can provide an early response indicator in patients with anal squamous cell carcinoma (ASCC) undergoing definitive chemoradiotherapy (CRT). Methods: Since 2021, patients with ASCC undergoing CRT at 2 institutions were offered ctDNA monitoring with the Natera Signatera assay, a commercial tumor-bespoke, multiplex PCR assay. All patients provided written informed consent for ctDNA testing. Patients were clinically restaged 3-4 months after CRT by clinical exam, endoscopy, and/or MRI, as well as annually with a chest, abdomen, and pelvis CT. Complete clinical response (cCR) was defined as having no tumor observed by digital exam, endoscopy, and/or MRI. Molecular ctDNA response was described according to cCR, tumor recurrence, and survival. Results: From January 2021 to October 2022, 41 patients with ASCC treated with CRT underwent ctDNA response assessment. Most patients (66%) had stage III disease. Patients were treated to a median radiation dose of 54 Gy in 27 fractions — with combinatorial mitomycin and fluoropyrimidine-based chemotherapy in 88% of patients, and fluoropyrimidine-based chemotherapy alone in 12%. The median follow-up was 22 weeks (range 0-89 weeks). ctDNA testing was performed in 36 patients at baseline, 31 patients during CRT, 27 patients within 40 days after CRT, 23 patients 3-6 months post-CRT, 23 patients 6-12 months post CRT, and 10 patients > 12 months post CRT. At baseline, 89% of patients had detectable ctDNA. Patients with stage III, as compared to stage I-II, disease had numerically higher baseline ctDNA levels (29 vs. 2.9 mean tumor molecules per milliliter (MTM/mL), p = 0.04). ctDNA levels decreased with treatment (24 vs. 2.1 MTM/mL, p = 0.005) among the 24 patients with detectable baseline ctDNA and ctDNA tested during CRT. Fifty eight percent of patients converted from ctDNA positive to ctDNA negative during CRT. Similarly, post-CRT ctDNA levels decreased (23 vs. 0.01 MTM/mL, p = 0.01), with 95% of patients converting from ctDNA positive to ctDNA negative. The time to ctDNA clearance was significantly shorter than the time to cCR (median 31 vs. 131 days, p < 0.0001). In follow up 2 patients reverted from ctDNA negative to ctDNA positive at 113 -155 days post-CRT. Currently all patients are clinically and radiographically without evidence of disease. Conclusions: Surveillance ctDNA monitoring provides an earlier response assessment for patients with ASCC undergoing CRT. However, longer term follow-up is required to determine if ctDNA response correlates with long-term recurrence free survival. Prospective trials are needed to assess the clinical utility of integrating molecular ctDNA response into therapeutic response surveillance.