Kinetoplastid parasites are early-diverged protozoans responsible for multiple diseases in humans. The entire genome of kinetoplastids is unusually organized into gene clusters containing functionally unrelated genes that are transcribed polycistronically by RNA Polymerase (RNAP) II. This genome arrangement has led to the assumption that these early-diverged eukaryotes lack RNAP II transcriptional regulation and instead regulate gene expression solely through post-transcriptional mechanisms. However, several chromatin modifications are enriched in the regions flanking gene clusters, including post-translationally modified histones, histone variants, and the DNA modification base J, suggesting that chromatin modifications impact the process of transcription in kinetoplastids. Here, two chromatin modifications enriched at transcription termination sites, base J and histone variant H3.V, and their effect on transcription and gene expression were investigated in the kinetoplastid species Trypanosoma brucei and Leishmania major. In T. brucei, we find that both J and H3.V independently promote transcription termination within gene clusters, thereby repressing the expression of genes located near the end of clusters. In L. major, base J has a more extensive role in promoting termination, where the loss of J between convergently transcribed gene clusters results in read through transcription and the production of antisense RNAs. Interestingly, this production of antisense RNAs does not negatively affect the abundance of sense mRNAs. J also promotes termination prior to the end of gene clusters in L. major, indicating that this function of J is conserved between L. major and T. brucei. Conversely, H3.V does not promote termination in L. major, despite its enrichment at termination sites. These findings provide the first evidence of RNAP II transcriptional regulation by chromatin modifications in kinetoplastids, specifically the promotion of termination within gene clusters to repress gene expression.