As global temperatures increase, understanding the genetic underpinnings of thermotolerance in heat-tolerant species is essential in order to optimize outcomes in crops and other important plant species. Prior studies have shown that several species in the genus Boechera can survive longer at higher temperatures than the model species A. thaliana. However, previous research into novel gene expression in two Boechera species, Boechera arcuata and Boechera perennans, has been constrained by lack of a sequenced genome in these species. In cases like this, RNA-Seq provides unique insight into gene expression. In this study, a bioinformatics pipeline was developed to analyze RNA-Seq data. Seedlings of B. arcuata and B. perennans were subjected to heat stress at 42°C for 3 hours. De novo transcriptomes for the control and experimental groups were assembled using Trinity. The original reads were re-aligned to the assembled transcriptomes to quantify the expression of each transcript using Bowtie2 and RSEM. After quantification, genes were analyzed for differential expression using the R package DESeq2. Upregulated and downregulated gene lists were defined and analyzed using GO term enrichment and hierarchical gene clustering. This analysis found that heat shock proteins made up the largest proportion of the upregulated genes, and GO terms related to the heat stress response were enriched in both species. However, heat stress transcription factors were more upregulated in B. arcuata than B. perennans. Similarly, only B. arcuata had enriched GO terms related to transcriptional regulation and promoter binding. Also, although ascorbate peroxidase (APX) genes are highly activated in A. thaliana during secondary reactive oxygen species (ROS) stress, APX was lowly upregulated in B. arcuata and not seen in B. perennans. Patterns in downregulated genes were very different between the two species. Like A. thaliana, B. perennans decreased expression of disease resistance genes and auxin-responsive genes. However, the dominant downregulated process in B. arcuata was cell wall metabolism, as seen in both enriched GO terms and in the downregulated gene cluster for this species. Additional research into the ROS response and the downregulation of cell wall metabolism is needed to further define mechanisms of thermotolerance in these species.