The overall classification of spidroins shown here agrees with previous reports. The poorer repertoire of spidroins found in the Mygalomorphae clade suggests. However, a MaSp2like gene has been found in the Mygalomorphae spider Avicularia juruenses. This spidroin was formerly considered an orbicularian synapomorphy, and provided evidence that spidroin paralogization occurred prior to the divergence of mygalomorph and araneomorph spiders, estimated at 240 million years ago. We used the presence of MaSps in Mygalomorphae as evidence that gene duplication occurred before the anatomical specialization of glands. Regarding the evolution of spidroin gene families, our phylogeny suggests that pyriform spidroins are the sister group of the flagelliform spidroins and that aciniforms had a more recent genetic ancestor with the tubulliform spidroins. The poor bootstrap values observed the basis of the tree may indicate that most spidroin gene families originated in an almost simultaneous event of gene duplication that probably occurred after the separation between Mygalomorphae and the other spider clades. This event resulted in further subfunctionalization and neofunctionalization of spidroin gene families in some organisms and allowed the creation and usage of different sorts of silk, some more resistant and others more flexible. For example, MaSp1 presents a number of poly-A residues that give the silk more resistance. This spidroin is mainly used to build the first radial sustentation of webs. However, the MaSp2 and MiSp spidroins contain GPG and GGS motifs that give the web its elastic and sticky properties; this type of silk is used to fill in the radial parts of the web. The presence or absence of amino acid motifs in spider silk may arise from different motif duplications and reorganizations in the genome, allowing this sort of molecular neofunctionalization. Once they have occurred, these rearrangements may give a specific mechanical property to the web and allow the accomplishment of different tasks in a spider’s life. By translating the spidroin coding sequences from contigs and singlets into proteins, we detected the presence of insertions and deletions in their nucleotide sequences. We assumed that they had been added by problems in sequencing or base-calling procedures probably caused by the repetitive molecular nature of spidroins. However, it is likely that some spidroin genes will be revealed to have degenerated to pseudo-genes after genome duplications, as reported in other cases. In contrast, some of these duplicated genes have probably acquired new functions; a broad study of their duplication, neofunctionalization, subfunctionalization and decay associated with pleiotropy, fitness and mutational trade-offs would produce an interesting molecular evolutionary story. Here, a comprehensive transcriptomic analysis was conducted for the first time to evaluate the gene expression content of Folinic acid calcium salt pentahydrate spinning glands from two evolutionary distant spiders. The number of sequences Catharanthine sulfate evaluated in this study was more than 2.5 times larger than all the Araneae data previously deposited into the dbEST database. The sheer size of our dataset attests to the efficiency of NGS strategies for gene discovery projects, even for organisms lacking genomic information. We were surprised that the CAP3 software, with its simplistic method for sequence assembly developed over 10 years ago, produced a better performance in EST clustering than more recently developed and updated software.