The complex physiology of 'Hass' avocado renders its postharvest ripening heterogeneous and unpredictable. Several approaches have previously been undertaken to broaden our understanding of the causes of this postharvest ripening heterogeneity but without much success. In this study, a fruit biopsy methodology was undertaken to sample mesocarp tissue from a series of individual avocado fruit while following individual fruit postharvest ripening characteristics without significantly disturbing their metabolism. Using both targeted and untargeted metabolomics approaches, we analyzed the metabolite profiles of the biopsies in order to get more insight into the biochemical mechanisms underlying 'Hass' avocado ripening heterogeneity. While C7 sugars (mannoheptulose and perseitol), dry matter and total Ca2+ were not correlated with time to reach edible ripeness, untargeted metabolomics profiling of polar and semi-polar compounds (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 to be differentially accumulating. In general, slowest ripening avocados had lower amounts of precursors of metabolites involved in key metabolic pathways. Our study indicates that comprehensive metabolomics may provide new markers for avocado ripening stage at harvest, and may give more insight into the complex ripening physiology of this fruit.