Abstract Purpose We aimed to use magnetic resonance imaging (MRI) to determine the relationship between the pathological depth of invasion (DOI), undetectability, and tumor thickness of squamous cell carcinoma of the floor of the mouth. Materials and methods We retrospectively evaluated the relationship between pathological DOI and MRI detectability, as well as the relationship between pathological DOI and tumor thickness on coronal fat-suppressed contrast-enhanced T1-weighted imaging or coronal T2-weighted imaging. Results We analyzed 30 patients with squamous cell carcinoma of the floor of the mouth; MRI revealed that the pathological DOI of the 11 undetectable lesions (median 2 mm) was smaller than that of the 19 detectable lesions (median 14 mm) (p < 0.001), and the cut-off value was 3 mm (sensitivity, 0.84; specificity, 0.91; area under the curve, 0.89). Tumor thickness on coronal fat-suppressed contrast-enhanced T1-weighted imaging was assessed in all 19 detectable lesions; however, tumor thickness on coronal T2-weighted imaging could not be assessed in eight cases. Tumor thickness on coronal fat-suppressed contrast-enhanced T1-weighted imaging was found to be significantly associated with the pathological DOI. Conclusions Undetectability on MRI indicates superficial lesions with a pathological DOI value that is less than 3 mm. In detectable lesions, tumor thickness on coronal fat-suppressed contrast-enhanced T1-weighted imaging is associated with pathological DOI.