The aim of this study was to analyze the physical and chemical characteristics of the maturation process of pitaya fruit (Hylocereus undatus) to identify indicators that can be used to determine the point of physiological maturity and establish the optimal timing of physiological maturity for harvesting the fruit. A completely randomized experimental design was employed and four biological repeats were performed. Physiological maturity was assessed using various physical characteristics: longitudinal length (LL), equatorial diameter (ED), pericarp thickness (PeT), pulp thickness (PuT), fruit mass (FM), pulp mass (PuM), pericarp mass (PeM), pericarp percentage (%Pe), pulp percentage (%Pu), pulp/pericarp ratio (Pu/Pe), pericarp color index (CI), hue color angle (h°), lightness index (L*), chroma (C*), blue-yellow variation (b*), and green-red variation (a*). Additionally, chemical characteristics such as soluble solid content (SS), titratable acidity (TA), SS/TA ratio, and pH were screened. The data were statistically analyzed by fitting regression models and computing Pearson's correlation coefficients (P < 0.05). Physiological maturity in pitaya fruits occurred between the 30th and 32nd days after anthesis, and this proved to be the optimal period for harvest. At this time, the fruit was completely red with high SS, and had the recommended values of TA, pH, and SS/TA ratio. During this period, ED, PuT, FM, PuM, %Pu, and Pu/Pe increased while PeT, PeM, and %Pe fell; these changes are considered desirable by producers and/or consumers. PuM was the variable that displayed more strong's association with other variables in the analysis.