Introduction: The lack of papillary structures and faint and/or unclear core features of follicular variant of papillary thyroid carcinoma (FV-PTC) may hamper the definitive fine needle aspiration biopsy -based diagnosis. Recently, the nomenclature of noninvasive encapsulated FV-PTC was revised to "noninvasive follicular thyroid neoplasms with papillary-like nuclear features" (NIFTP). However, it remains inconclusive whether or not the peptide patterns differ between NIFTP and encapsulated FV-PTC. The main objectives of this study were to investigate the viability of matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) in the pathological assessment of NIFTP and to evaluate the discriminatory power of MALDI MSI for the classification of classical variant of PTC (CV-PTC), NIFTP, and encapsulated FV-PTC. Methods: MALDI MSI was employed to investigate the changes in peptide profiles from 21 formalin-fixed paraffin-embedded (FFPE) tissue samples (n = 7 from each group of CV-PTC, NIFTP, and FV-PTC). Six out of seven FV-PTC FFPE tissue samples were encapsulated FV-PTC; only one was infiltrative FV-PTC. Liquid chromatography-tandem mass spectrometry was used for the identification of the peptide signals detected in MALDI MSI. Results: Using receiver operating characteristics analysis, 10 peptide signals distinguished NIFTP from normal thyroid parenchyma (area under the curve [AUC] >0.80). To evaluate the discriminatory power of MALDI MSI, statistically significant peptide signals (n = 88) within three groups were used for hierarchical clustering. The method had high discriminatory power for distinguishing CV-PTC from NIFTP and FV-PTC (encapsulated and infiltrative). The majority of the NIFTP and encapsulated FV-PTC were clustered together, indicating that NIFTP could not be distinguished from encapsulated FV-PTC. However, infiltrative FV-PTC FFPE tissue samples had the furthest distance from all the NIFTP cases. High signal intensities of S100-A6, vimentin, and cytoplasmic actin 1 were detected in FV-PTC, prelamin A/C in CV-PTC, and 60S ribosomal protein L6 and L8 in NIFTP tissues. Conclusions: MALDI MSI, a powerful tool combining histological and mass spectrometric data, enabled the differentiation of NIFTP from normal thyroid parenchyma. Although NIFTP is a recent definition that replaces noninvasive encapsulated FV-PTC, the peptide profiles of NIFTP and encapsulated FV-PTC were found to be similar.