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Conventional methods for breast tumour margins assessment need a long turnaround time, which may lead to re‐operation for patients undergoing lumpectomy surgeries. Photoacoustic tomography (PAT) has been shown to visualize adipose tissue in small animals and human breast. Here, we demonstrate a customized multimodal ultrasound and PAT system for intra‐operative breast tumour margins assessment using fresh lumpectomy specimens from 66 patients. The system provides the margin status of the entire excised tissue within 10 min. By subjective reading of three researchers, the results show 85.7% [95% confidence interval (CI), 42.0–99.2] sensitivity and 84.6% (95% CI, 53.7–97.3) specificity, 71.4% (95% CI, 30.3–94.9) sensitivity and 92.3% (95% CI, 62.1–99.6) specificity and 100% (95% CI, 56.1–100) sensitivity and 53.9% (95% CI, 26.1–79.6) specificity, respectively, when cross‐correlated with …