The complex physiology of ‘Hass’ avocado renders its postharvest ripening heterogeneous and unpre-dictable. Several approaches have previously been undertaken to broaden our understanding of the causesof this postharvest ripening heterogeneity but without much success. In this study, a fruit biopsy method-ology was undertaken to sample mesocarp tissue from a series of individual avocado fruit while followingindividual fruit postharvest ripening characteristics without significantly disturbing their metabolism.Using both targeted and untargeted metabolomics approaches, we analyzed the metabolite profiles of thebiopsies in order to get more insight into the biochemical mechanisms underlying ‘Hass’ avocado ripen-ing heterogeneity. While C7sugars (mannoheptulose and perseitol), dry matter and total Ca2+were notcorrelated with time to reach edible ripeness, untargeted metabolomics profiling of polar and semi-polarcompounds (based on GC–MS and LC–MS platforms), revealed several metabolites, mainly amino acids,that were related to ripening heterogeneity. In addition, analysis of fatty acids revealed linoleic acid tobe differentially accumulating. In general, slowest ripening avocados had lower amounts of precursors ofmetabolites involved in key metabolic pathways. Our study indicates that comprehensive metabolomicsmay provide new markers for avocado ripening stage at harvest, and may give more insight into thecomplex ripening physiology of this fruit.