Ultimately for some groups of fungi there may need to be two barcodes to meet the requirements of identifying very closely related fungal species. In order to develop a suitable barcode system for Fusarium species complexes, universally accepted fungal barcode, Internal Transcribed Spacer (ITS, 600bp) was examined for the barcoding performance with other potential barcode loci viz., Cytochrome Oxidase subunit I (COI, 567bp) and Translation elongation factor 1 (tef-I, 700 bp). One novel genomic region (NADH dehydrogenase subunit 6 (ND6), 900bp) was also be evaluated. Multilocus sequence Typing (MLST) of 22 Fusarium isolates representing eleven different species (F. acuminatum, F. chlamydosporum, F. graminearum, F. oxysporum, F. pallidoroseum F. poae, F. solani, F. sporotrichioides, F. subglutinans, F. udum and F. verticillioides), were compared. The ITS regions did not work well within the Fusarium species complex, and its near relatives due to less barcode gap (0.12). Comparative sequence analyses suggested COI to be a better marker for intraspecific and interspecific variability because of greater barcode gap (0.50), Transition/Transversion ratio (1.48) and evolutionary divergence (0.49).  However, the presence of mobile introns and low PCR success in COI region poses a serious difficulty in the PCR and bioinformatic surveys. Therefore, ND6 could be considered as a suitable secondary barcode candidate for Fusarium species complex due to the absence of introns, justified barcode gap (0.48), higher Transition/Transversion ratio (1.42) and evolutionary divergence (0.44).