Genome annotation is a set of algorithmic software tools that allows complete sequencing of an organism’s genome. Kytococcus sedentarius is a gram positive bacterium, with callus degrading activity, closely associated with pitted keratolysis. The objective of this study was to annotate the genes in Kytococcus sedentarius strain 541 (DSM 20547) responsible for heme biosynthesis, involving the formation of uroporphyrinogen III from L-Glutamate. Metabolic functions of individual genes were characterized using the Integrated Microbial Genome Annotation Collaboration Toolkit (IMG-ACT) developed by the Joint Genome Institute (University of California, CA). Sequence based similarity was identified using BLAST and T-COFFEE. Cellular localization was determined using TMHMM and SignalP. Functional amino acids were assessed using WebLogo and protein functions were analyzed using Pfam. TIGRFAM was used to gather structure based evidence. KEGG and MetaCyc were used to verify metabolic functions of respective genes in the pathway. Significant importance was also given to the novel open reading frames, conserved domains, gene duplication, degradation and phylogenetic evolution. Genes were localized mostly in the cytoplasmic region and Weblogo indicated highly conserved amino acids. The proposed DNA coordinates could not be verified for glutamyl tRNA reductase (gene object id: 644992667). There was no evidence of any gene being a pseudo gene and phylogenetic analysis did not indicate any horizontal gene transfer. Heme is important in a number of microbial metabolic processes. Annotation reports indicated that the genes leading to the synthesis of uroporphyrinogen III were functionally active and highly conserved in Kytococcus sedentarius.