Diapause is a physiological state of developmental arrest in organisms adapted to respond to predictable unfavorable environmental changes. Adult females of Tetranychus urticae Koch (Tetranychidae) undergo facultative reproductive diapause only if they were exposed to environmental diapause stimuli (short days and low temperatures) during their immature stages. This response is commonly reported in temperate regions. Recently, T. urticae females displaying diapause phenotype (red instead of a greenish color and cessation of oviposition) were found for the first time in populations infesting papaya plants in the states of Bahia and Espírito Santo, Brazil. The tropical climate common to these states does not provide the environmental cues typically required for diapause induction even during winter. This study aimed to mine transcriptomes of non-diapausing and diapausing phenotypes of T. urticae to provide a molecular characterization of diapause in a tropical population of T. urticae by performing comparative transcriptional activity between diapausing and non-diapausing mites. Twenty female mites sampled from papaya plants in the state of Espírito Santo were placed on an arena made of jack bean leaf with the abaxial surface upwards, positioned on a moistened sponge, and surrounded by moistened cotton wool to prevent escape. A total of four arenas were established and maintained under short day and low temperature conditions (15 ± 1 °C, 10:14 h L:D), a standard protocol sufficient to induce diapause in this species, until the progeny reached adulthood. Diapausing (red, non-ovipositing) and non-diapausing (green, ovipositing) adult female progeny were subjected to RNA extraction, cDNA library construction, and Illumina sequencing. Reads were mapped and counted against a T. urticae reference genome. The experiment consisted of two treatments (diapausing and non-diapausing females), with six biological replicates per treatment, each containing a pool of 25 mites. A total of 14,185 genes were identified, including 601 differentially expressed genes (DEGs), of which 488 were up-regulated and 113 were down-regulated. After enrichment analysis, DEG transcripts associated with pathways including cytoskeleton in muscle cells (adj. p = 0.027) and metabolism of xenobiotics by cytochrome P450 (adj. p = 0.027) were up-regulated, while those associated with DNA replication (adj. p = 0.006) and protein processing in the endoplasmic reticulum (adj. p = 0.006) were down-regulated. Future characterization of pathways and genes associated with diapause phenotype will allow the understanding of the molecular processes underlying this phenotype in T. urticae.