RNA contains over 100 post-transcriptional chemical modifications. Of these, the most abundant is pseudouridine (Ψ), the 5’ ribosyl isomer of uridine. The formation of Ψ occurs at the polynucleotide level, and is catalyzed by Ψ synthase enzymes. To date, five families of Ψ synthases have been identified. Our work deals with the fifth and last family to be discovered, the Ψ synthase TruD family. TruD homologs are present in organisms across the three kingdoms of life. A sequence alignment of TruD homologs reveals distinct sequence insertions at specific and conserved locations in homologs from higher organisms. We have carried out extensive bioinformatics searches in an effort to characterize these insertion sequences, and have found that one of these insertions has a high degree of similarity to the R3H single-strand nucleic acid binding domain. To further understand the nature of this insertion, we examined its role in the enzymatic activity of the yeast TruD homolog, Pus7p, and found that mutating this insert decreased the enzyme’s activity by almost half. The human genome codes for two TruD homologs, PUS7 and PUS7L. These putative pseudouridine synthases were named based on their similarity to the yeast TruD homolog Pus7p, but their function has only been inferred based on sequence, and neither their activity, nor their structure have been examined. In an effort to further our understanding about the TruD synthase family, we have taken a closer look at PUS7 and PUS7L. We have determined the optimal conditions for over-expression of the enzymes in a bacterial expression system, and shown that the proteins are soluble, and can be purified using serial chromatography. In addition, we have queried these enzymes’ ability to recognize canonical TruD substrates and found that they are unable to complement an E. coli truD deletion or an S. cerevisiae pus7 deletion. Instead, the human PUS7 enzyme proved to be highly specific to the human tRNAGlu sequence, displaying activity targeted to U13. This activity appears to be independent of accessory factors. Taken together, this work strives to further our knowledge of Ψ synthases by examining TruD homologs present in higher organisms. TruD has the least sequence identity to the other four synthase families and does not possess any of the known RNA binding motifs. This work expands our knowledge base of the TruD family, the most divergent of the five Ψ synthase families.