Hepatocellular lipid accumulation characterizes nonalcoholic fatty liver disease (NAFLD). However, the types of lipids associated with disease progression are debated, as is the impact of their localization. Traditional lipidomics analysis using liver homogenates or plasma dilutes and averages lipid concentrations, and does not provide spatial information about lipid distribution. We aimed to characterize the distribution of specific lipid species related to NAFLD severity by performing label-free molecular analysis by mass spectrometry imaging (MSI). Fresh frozen liver biopsies from obese subjects undergoing bariatric surgery ( n = 23) with various degrees of NAFLD were cryosectioned and analyzed by matrix-assisted laser desorption/ionization (MALDI)-MSI. Molecular identification was verified by tandem MS. Tissue sections were histopathologically stained, annotated according to the Kleiner classification, and coregistered with the MSI data set. Lipid pathway analysis was performed and linked to local proteome networks. Spatially resolved lipid profiles showed pronounced differences between nonsteatotic and steatotic tissues. Lipid identification and network analyses revealed phosphatidylinositols and arachidonic acid metabolism in nonsteatotic regions, whereas low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL) metabolism was associated with steatotic tissue. Supervised and unsupervised discriminant analysis using lipid based classifiers outperformed simulated analysis of liver tissue homogenates in predicting steatosis severity. We conclude that lipid composition of steatotic and nonsteatotic tissue is highly distinct, implying that spatial context is important for understanding the mechanisms of lipid accumulation in NAFLD. MSI combined with principal component-linear discriminant analysis linking lipid and protein pathways represents a novel tool enabling detailed, comprehensive studies of the heterogeneity of NAFLD.