The aspergilli comprise a diverse group of filamentous fungi spanning over 200 million years of evolution. In this work, we report a clarification of the phylogenetic relationship between A. carbonarius and A. tubingensis by the screening of ITS-5.8RDNA, (niiA-niaD), ß-tubulin and EF-1 genes. Phylogenetic analysis of aspergilli ITS-5.8rDNA sequences divided A. carbonarius and A. tubingensis into two coherent clusters and showed a close intergeneric relationship which is in keeping with the existing morphological and taxonomic classification. Herein, A. carbonarius exhibit a complete similarity (100%) for the three target genes: intergenic region of (niiA-niaD), ß-tubulin and EF-1. Tree resulting from a bootstrap analysis indicate branch points when the significance is 98% minimum for each gene (ß-tubulin and EF-1). The phylogenetic analysis of aspergilli genome structure provided a quantitative evaluation of forces driving long-term eukaryotic genome evolution. It also led to an experimentally validated model of mating-type locus evolution, suggesting the potential for sexual reproduction in A. carbonarius and A. tubingensis. This is the first work describing evolutionary relationship between both species. At the same time, this finding can be one of the natural processes in the course of evolution of new taxa.