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The genus <i>Fusarium</i> comprises more than 300 species, and many of them are pathogens that cause severe diseases in agricultural, horticultural and forestry plants in both antropogenic and natural ecosystems. Because of their importance as plant pathogens, the genomes of several <i>Fusarium</i> spp. have been sequenced. Within this genus, <i>Fusarium circinatum</i> is one of the most harmful pathogens of pine trees attacking up to 60 <i>Pinus</i> species. Till now, the genomes of 13 strains of <i>F. circinatum</i> have been sequenced. The strain GL1327 we studied lacks a twelfth chromosome, which allows the study of virulence genes on this chromosome. Although the genome of several strains of <i>F. circinatum</i> has been sequenced, it is still almost completely unannotated, which severely limits the possibilities to further investigate the molecular mechanisms of virulence of <i>Fusarium</i>. Therefore, this study aimed to annotate the 12th chromosome of <i>F. circinatum</i> and integrate currently available resources. In silico annotation of the 12th chromosome of <i>F. circinatum</i> revealed the presence of 118 open reading frames (ORFs) encoding 141 proteins which were predicted using an ab initio gene prediction tool. The InterProScan and SMART analyses identified known domains in 30 proteins and eggNOG additionally in 12 of them. Among them, four groups can be distinguished: genes possibly related to heterokaryon incompatibility (4 genes), regulation of transcription (5 genes), plant cell wall degrading enzymes (7 genes) and trichothecene synthesis (3 genes). This study also integrated data of <i>F.circinatum</i> reference strain CMWF1803 assembled to chromosome level but not annotated with currently best annotated but assembled only to scaffold level strain NRRL 25331.