Maturation is a critical developmental process, and the age and size at which it occurs have important fitness consequences. Although maturation is remarkably variable, certain mechanisms, including a minimum size or state threshold, are proposed to underlie the process across a broad diversity of taxa. Recent evidence suggests that thresholds may themselves be developmentally plastic, and in the crustacean Daphnia pulex it is unclear whether maturation follows a threshold or is a gradual process more akin to a rate. Changes in gene expression across four instars before and during maturation were compared in a cDNA microarray experiment. Developmental stage was treated statistically both as a discontinuous and as a continuous variable, to determine whether genes showed gradual or discrete changes in expression. The continuous analysis identified a greater number of genes with significant differential expression (45) than the discontinuous analysis (11). The majority of genes, including those coding for histones, factors relating to transcription and cell cycle processes, and a putative developmental hormone showed continuous increases or decreases in expression from the first to the fourth instars that were studied, suggestive of a prolonged and gradual maturation process. Three genes coding for a fused vitellogenin/superoxide dismutase showed increases in expression following the second instar and coincided with the posited maturation threshold, but even their expression increased in a continuous fashion.