Damage to the genetic material of the cell poses a universal threat to all forms of life. The DNA damage response is a coordinated cellular response to a DNA break, key to which is the phosphorylation signaling cascade. Identifying which proteins are phosphorylated is therefore crucial to understanding the mechanisms that underlie it. We have used stable isotopic labeling of amino acids in cell culture-based quantitative phosphoproteomics to profile changes in phosphorylation site abundance following double stranded DNA breaks, at two distinct loci in the genome of the single cell eukaryote Trypanosoma brucei. Here, we report on the T. brucei phosphoproteome following a single double-strand break at either a chromosome internal or subtelomeric locus, specifically the bloodstream form expression site. We detected >6500 phosphorylation sites, of which 211 form a core set of double-strand break responsive phosphorylation sites. Along with phosphorylation of canonical DNA damage factors, we have identified two novel phosphorylation events on histone H2A and found that in response to a chromosome internal break, proteins are predominantly phosphorylated, while a greater proportion of proteins dephosphorylated following a DNA break at a subtelomeric bloodstream form expression site. Our data represent the first DNA damage phosphoproteome and provides novel insights into repair at distinct chromosomal contexts in T. brucei.