Evidence-based comparative genomics was used to annotate contig 53 genomic sequence spanning 30,500 bp on the dot chromosome of Drosophila ficusphila. The project was completed with the help of the Genomics Education Partnership undergraduate student research initiative. In Drosophila the distal portion of the dot chromosome is of interest due to having heterochromatic characteristics but maintains a gene density similar to euchromatic regions. The databases and web‐based tools of FlyBase, GEP UCSC Genome Browser Mirror, NCBI BLAST, Gene Record Finder, and Small Exon Finder were used for gene annotation of this region. The genomic DNA sequence was compared with the Drosophila melanogaster genome and five putative orthologs were annotated: CG33521, PIP4k, Mitf, Arf102F, and Dyrk3. These genes showed conservation with D. melanogaster genes, ranging between 64.1%‐98.3% identity (%ID) conservation. Genes known to have many biologically crucial functions, such as Arf102F (%ID=98.3) and PIP4K (%ID=97.5), showed high amino acid sequence similarity. Mitf (%ID=74) and Dyrk (%ID=75.9) have less biologically important roles within the organism and showed lower similarity. Genes identified as not having biologically crucial functions, such as CG33521 (%ID=64.1), showed very low sequence similarity between the two species. CG33521 gene codes for a Zinc ion binding protein. PIP4K gene codes for a protein kinase involved in many pathways including cell growth, cell adhesion, and actin filament organization. Mitf gene codes for a transcription factor involved in the regulation of protein dimerization. Dyrk gene codes for a protein kinase involved in phosphorylating ATP-binding proteins. These findings will help contribute a better understanding of the characteristics and evolution of the dot chromosome within Drosophila.