Fruit of pepper (Capsicum annuum L.) is hollow by nature, which limits its water reservoir capacity, and as such, small amounts of water loss result in loss of freshness and firmness, which reduce fruit quality, shelf life, and market value. In order to understand the basis for water loss from fruit, 10 pepper accessions with wide variation in water loss rate were used to study physiological and biochemical factors associated with postharvest water loss in ripe pepper fruit during storage. Postharvest water loss rate in ripe pepper fruit stored at 20 °C, and 85% relative humidity, was found to be associated with cell membrane ion leakage, lipoxygenase activity, and total cuticular wax amount. Total cuticular wax amounts were highest in the high-water-loss pepper fruit, and lowest in the low-water-loss fruit. However, total cuticle amount (isolated enzymatically and quantified gravimetrically), total cutin monomer amount, and the amount of individual cutin monomer and wax constituents (determined using gas chromatography mass spectrometry) indicated no direct association with postharvest water loss rates. Fruit fresh weight, pericarp weight, pericarp surface area, pericarp thickness, initial water content, and dry matter were highly associated with each other, but less so with water loss rate. Fruit of accessions displaying high fruit water loss rate matured and ripened earlier than fruit of accessions displaying low-water-loss rate. Cell membrane ion leakage and lipoxygenase activity were higher after storage than immediately after harvest. Pepper fruit total cuticle wax amount, lipoxygenase activity, and cell membrane ion leakage were directly related to postharvest water loss rate in pepper fruit during storage.