Keloid disease (KD) is a quasineoplastic fibroproliferative tumour of unknown origin causing a progressive, recurrent dermal lesion. KD is not homogeneous in nature and shows phenotypic structural differences between its distinct peripheral margins compared to its centre. The keloid margin is often symptomatically more active with increased dermal cellularity, compared to a symptomatically dormant and hypocellular centre of lesion. The aim of this study was to delineate the morphological components of a keloid scar tissue by measuring the differences between various anatomical locations within the keloid tissue, such as the margin and the centre of the lesion compared to its surrounding normal skin using Fourier transform infrared (FT-IR) microspectroscopy. FT-IR microspectroscopy is a technique that produces spectra with detailed molecular biochemical information inherent of the chemical structure. Chemical maps were constructed on extralesional cross sections taken from six keloid scars. H&E stained sections were used to confirm diagnosis of keloid and orientate the experimental cross sections prior to FT-IR. Spectral band assignment and principal components analysis were conducted. Distinct vibrational bands (100 spectra) were observed using FT-IR spectroscopy. Partial least squares discriminant analysis, with bootstrapping (10,000 analyses), identified whether a spectrum was from the keloid or normal tissue showing an average accuracy of 84.8%, precision of 80.4%, specificity of 76.2%, and sensitivity of 92.9%. FT-IR microspectroscopy showed significant differences in spectral profiles in keloid tissue in different anatomical locations within the cross section. We believe that this proof-of-concept study may help substantiate the use of FTIR spectroscopy in keloid diagnosis and prognosis.