Multiplex 1 consists of eight SNPs and allows detection of the more frequent major clades in Europe. It also includes the 12f2 deletion, which determines haplogroup J and was typed by presence/absence in the PCR amplification (Figure 2). Multiplex 1 determines haplogroups R1b1-P25, R1a1-SRY1532 and I2a-P37b. We performed multiplex 1 in all samples, and depending on the results, multiplex 2, 3, 4 or 5 was performed in order to define the haplogroup more precisely.

Multiplex 2 comprises four SNPs, multiplex 3 and 4 comprise six SNPs each, while multiplex 5 is a combination of five SNPs. Multiplex 2 subdivides haplogroup I, multiplex 3 haplogroup E, multiplex 4 haplogroup J, while haplogroup 5 determines haplogroups G-M201, H-M69, L-M22, N1c-Tat and T-M70.

Among the DNA samples we studied, we detected 20 different Y haplogroups, of which five haplogroups (E1b1b1a-M78, I2a-P37b, J2b2-M241, R1a1-SRY1532 and R1b1-P25) comprised 72.6% of the studied Y chromosomes (Table 3). The distribution of the Y haplogroups in Macedonians, Albanians and males of other ethnic origin (Roma, Serbs and Turks) is given in Table 3. The most common Y haplogroup in Macedonians was I2a-P37b (27.5%), which was followed by E1b1b1a-M78 (15.6%), R1a1-SRY1532 (14.2%) and R1b1-P25 (11.4%). In the Albanians, E1b1b1a-M78 was found in 28.8%, R1b1-P25 in 18.0%, J2b2-M241 in 13.5% and R1a1-SRY1532 in 12.6%. In the small group of Roma, Serbs or Turks, R1b1-P25 was found in 23.8% and E1b1b1a-M78 and H-M69 in 14,3% each. All three males with haplogroup H-M69 were of Roma ethnic origin.