The assessment of the transformation zone is a critical step toward diagnosis of cervical cancer. This work involves the development of a portable, label‐free transvaginal multispectral diffuse optical imaging (MDOI) imaging probe to estimate the transformation zone. The images were acquired from N = 5 (N = 1 normal, N = 2 premalignant, and N = 2 malignant) patients. Key parameters such as spectral contrast ratio (ρ) at 545 and 450 nm were higher in premalignant (0.29, 0.25 for 450 nm and 0.30, 0.17 for 545 nm) as compared to the normal patients (0.13 and 0.14 for 450 and 545 nm, respectively). The threshold for the spectral intensity ratio R610/R450 and R610/R545 can also be used as a marker to correlate with the new and original squamous columnar junction (SCJ), respectively. The pilot study highlights the use of new markers such as spectral contrast ratio (ρ) and spectral intensity ratio (R610/R450 and R610/R545) images.