Mammillaria bombycina is a cactus distributed in the central region of Mexico. This species is in danger of extinction, and its in vitro cultivation has, therefore, been established for reinsertion into its natural habitat. Due to this species’ relatively easy propagation, its use has been ideal for analysis at the gene level. In this contribution, a de novo transcriptome of M. bombycina was produced under in vitro conditions. The samples were sequenced using an Illumina platform, obtaining an average of 24 million clean readings. From the assembly and annotation, 84,975 transcripts were generated, 55% of which were unigenes. Among these, the presence of 13 isoforms of genes belonging to glyoxalase I, II, and III were identified. An analysis of qRTPCR expression of these genes was performed under in vitro and ex vitro conditions and dehydration at 6 and 24 h. The highest expression was observed under greenhouse conditions and dehydration at 24 h, according to the control. The de novo assembly of the M. bombycina transcriptome remains a study model for future work in cacti.