Colletotrichum causing anthracnose in mango is known for its variable virulence that may have an effect on disease development and efficacy of management strategies. In this study, we characterized Colletotrichum spp. isolated from mango fruits under in vitro and in vivo conditions using close-range thermography and reflectance spectroscopy. Twenty-six isolates were phylogenetically characterized to ascertain species using the internal transcribed spacer (ITS) sequence. Virulence, spectral (in vivo and in vitro), and thermographic responses (in vivo) of these isolates were analyzed. Isolates were grouped into the Colletotrichum gloeosporioides species complex and classified into eight morphotypes. Mycelial growth, conidia production, sporulation abundance, and area under disease progress curve (AUDPC) varied largely among isolates. Disease symptoms were observed 4 days after inoculation (dai), and for most morphotypes, changes in tissue temperature were registered at 11 dai, with the highest decrease at 14 dai with pathogen sporulation. In vitro and in vivo morphotypes shared changes in the spectrum range and main variations were found in the number of informative spectral bands. In vivo average gross reflectance was higher in disease-inoculated tissue than in healthy uninoculated tissue. Morphotype responses varied depending on AUDPC values and post-inoculation time. Discriminant analysis of the spectral response using Principal Component Analysis (PCA) and Partial Least Squares Regression (PLSR) explained 94-96.3 and 98-99.9% of the variance from in vitro and in vivo tests, respectively. Spectral markers were obtained for four distinct morphotype groups. We found three (550-650, 650.1-790, and 1300-1400 nm) and two (520-830, and 1100-1450 nm) regions with highly (P<0.05) discriminant spectral bands for diseased fruits and morphotype characterization.