We investigated the pattern of molecular variation with respect to secondary structure in the 16S ribosomal RNA gene and its phylogenetic implications for arachnids with a focus on spiders. Based on a model by Gutell et al. (1996), secondary structures were proposed for the 3′ half of 16S in the mygalomorph spider Aptostichus atomarius. Models were also constructed for a hypervariable length of the 16S in three other arachnids, which revealed a trend of stem and loop reduction in more advanced arachnids. Using a simple statistical approach to compare functional regions, we found that internal and external loops are more variable than stems or connection regions. Down-weighting or excluding regions which code for the more variable loops improved tree topologies by restoring the monophyly of the genus Aptostichus, a group supported by combined 16S, COI, and morphological data in other analyses. This study demonstrated the utility of considering secondary structure for DNA sequence alignment and phylogenetic reconstruction in spiders.