The precise and reliable identification of HLA alleles is critical in hematopoietic progenitor transplantation and also important in population studies and studies exploring the association of HLA alleles to certain diseases. In the past few years, the sequencing based typing approach (SBT) stood up as the most comprehensive method for HLA genotyping, providing the most detailed information deriving from the polymorphic loci. However, one of the inherent problems with this typing method is the interpretation of ambiguous allele combinations. In this study we have investigated the HLA*DRB1 locus in total of 360 volunteer donors using SBT and we have analyzed the occurrence of ambiguous results. The SBT for exon 2 of DRB1 gene has been performed using an automated DNA sequencer AlfExpress. After one round of SBT, 87/360 unambiguous results (24.2%) were obtained with differences in the fifth decimal position being taken into consideration. When eliminating differences in the fifth decimal and setting the resulting genotype to four decimals, the total number of unambiguous genotypings rose to 129/360 (35.8%). The ambiguities occur in all DRB1 families tested but most frequently involve the two most frequent HLA-DRB1 antigens in Macedonian population, HLA-DRB1*11 and HLA-DRB1*16. The most frequently found ambiguous combinations (present in more then 1% of the total cases) were: DRB1*0101/07 + DRB1*16011 (1.9%), DRB1*0101/07 + DRB1*03011 (1.4%), DRB1*03011/04 + DRB1*16011/08 (1.7%), DRB1*0101/07 + DRB1*1104 (1.4%), DRB1*11011/ 012/013/014/30/05 + DRB1*16011/012 (1.1%), DRB1*11014/011/041 + DRB1*11041/05/30 (1.1%) and DRB1*1114/05/20/041/02 + DRB1*1342/011/11/021/05 (1.1%). In the resolution of the ambiguous results gained after SBT, several approaches are available. In our laboratory, we are currently conducting studies aimed to determine the efficacy of the different procedures proposed for resolution of ambiguous results in order to determine the most adequate for the Macedonian population.