We explore diagnostic utility of a multicolor excitation multimodal nonlinear optical (NLO) microscopy for noninvasive detection of squamous epithelial precancer in vivo. The 7,12-dimenthylbenz(a)anthracene treated hamster cheek pouch was used as an animal model of carcinogenesis. The NLO microscope system employed was equipped with the ability to collect multiple tissue endogenous NLO signals such as two-photon excited fluorescence of keratin, nicotinamide adenine dinucleotide, collagen, and tryptophan, and second harmonic generation of collagen in spectral and time domains simultaneously. A total of 34 (11 controlled and 23 treated) Golden Syrian hamsters with 62 in vivo spatially distinct measurement sites were assessed in this study. High-resolution label-free NLO images were acquired from stratum corneum, stratum granulosum-stratum basale, and stroma for all tissue measurement sites. A total of nine and eight features from 745 and 600 nm excitation wavelengths, respectively, involving tissue structural and intrinsic biochemical properties were found to contain significant diagnostic information for precancers detection (). Particularly, 600 nm excited tryptophan fluorescence signals emanating from stratum corneum was revealed to provide remarkable diagnostic utility. Multivariate statistical techniques confirmed the integration of diagnostically significant features from multicolor excitation wavelengths yielded improved diagnostic accuracy as compared to using the individual wavelength alone.