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Abstract Background The protozoan pathogen Toxoplasma gondii has the unique ability to develop a chronic infection in the brain of its host by transitioning from the fast growing tachyzoite morphology to latent bradyzoite morphology. A hallmark of the bradyzoite is the development of neuronal cysts that are resilient against host immune response and current therapeutics. The bradyzoite parasites within the cyst have a carbohydrate and protein-rich wall and a slow-replication cycle, allowing them to remain hidden from the host. The intracellular, encysted lifestyle of T. gondii has made them recalcitrant to molecular analysis in vivo. Results Here, we detail the results from transcriptional and proteomic analyses of bradyzoite-enriched fractions isolated from mouse brains infected with T. gondii over a time course of 21 to 150 days. The enrichment procedure afforded consistent identification of over 2000 parasitic peptides from the mixed-organism sample, representing 366 T. gondii proteins at 28, 90, and 120 day timepoints. Deep sequencing of transcripts expressed during these three timepoints revealed that a subpopulation of genes that are transcriptionally expressed at a high level. Approximately one-third of these transcripts are more enriched during bradyzoite conditions compared to tachyzoites and approximately half are expressed at similar levels during each phase. The T. gondii transcript which increased the most over the course of chronic infection, sporoAMA1, shows stage specific isoform expression of the gene. Conclusions We have expanded the transcriptional profile of in vivo bradyzoites to 120 days post-infection and provided the first in vivo proteomic profile of T. gondii bradyzoites. The RNA sequencing depth of in vivo bradyzoite T. gondii was over 250-fold greater than previous reports and allowed us to identify low level transcripts and a novel bradyzoite-specific isoform of sporoAMA1.