Purpose: To understand time of day variation in Arabidopsis heat shock responses. We have observed significant differences in terms of plant survivability in response to a heat shock across various times of day. Because of this, we believe that transcriptional responses may be a significant underlying factor which contributes to the observed physiology and surivability after heat stress. Methods: Whole plant tissue mRNA profiles in Col-0 Arabidopsis were generated by deep sequencing using Illumina HiSeq 2500. Tissue samples of four replicates were collected at two times of day (Dawn and Dusk, ZT0/ZT12) and were either control or heat stressed (30oC) for 1 hour. An additional 4 samples were collected in constant light conditions (single replicate) to quantify circadian effects. Sequence reads from each sample were mapped to the TAIR10 reference genome with Tophat2 and counts were obtained using Htseq-count. Differential expression analysis was done using DeSeq2 to calculate differential expression levels of each gene in the dataset. Results: We identified 5512 differentially expressed transcripts with our DESeq2 based data analysis pipeline that involves characterizing gene expression profiles based on basal expression level, time of differential expression, and direction of differential expression. An additional interaction term analysis revealed 572 genes which showe time of day dependent responses to heat stress, meaning the response to heat stress differs depending on the time of day that the heat stress is perceived by the plant. Downstream analysis using gene ontology analysis tools (Pantherdb.org) and Cytoscape revealed strong differences in terms of response happening in the AM and PM gene expression networks. The significance of this work is that it is the first study to show that there is a significant difference in terms of transcriptional responses to heat stress in Arabidopsis where the differences were quantified by levels of mRNA expression. Overall design: Whole tissue mRNA profiles were measured by deep sequencing with four replicates in Col-0 ecotype Arabidopsis. Samples collected were control samples at two times of day (dawn and dusk) and heat stressed samples at two times of day (dawn and dusk). Additionally, 4 single replicate samples were collected in contant light conditions (two times of day/control and treatment) to quantify circadian effects. Reads were collected using Illumina Hiseq 2500 and analyzed with dowstream computational analysis.