Abstract: Physiological variability within a large canopy ‘d’Anjou’ tree results from agronomic and environmental factors. Fruit diversity within the canopy was surveyed using metabolic proﬁling to identify metabolism associated variability within the canopy. Different portions of the same fruit were evaluated to determine future precise sampling protocols for metabolic proﬁling of pear. We expected that the metabolic proﬁle of the peel and cortex would be diverse and these differences would highlight speciﬁc metabolic pathways as inﬂuenced by these conditions. Another focus of this work was developing an untargeted metabolic proﬁling protocol tailored for pear using a combination of extractions coupled with GC-MS and LC-MS analysis. ‘d’Anjou’ pear fruit harvested from two different zones of trees trained to an open vase canopy were maintained at room temperature for 24 days to observe any changes in external phenotype and metabolic proﬁle. Fruit harvested from the internal canopy were greener as also indicated by high Index of Absorbance Difference (IAD) and hue angle values. Metabolic proﬁle differences between tree positions were widespread and included metabolites from many pathways beyond those associated with peel color. In addition, peel metabolic proﬁle was different depending upon the tissue position (top vs. bottom) sampled from the pears. Speciﬁc pathways altered by tree position included those potentially linked to fruit quality and ripeness, including malic acid and aroma volatile (V) levels, as well as light environment, such as ﬂavonol glycoside levels. Present results warrant further future work targeting these changes over time during storage and alongside fruit quality analyses to validate the impacts on ripening and tree factors. In addition, outcomes indicate tissue sampling strategies require consistency with respect to the region of the pear fruit sampled for metabolomics.