<jats:title>Abstract</jats:title><jats:p>This is the first report of population variability of the contents of <jats:italic>n</jats:italic>‐alkanes and nonacosan‐10‐ol in the needle epicuticular waxes of Serbian spruce (<jats:italic>Picea omorika</jats:italic>). The hexane extracts of needle samples originated from three natural populations in Serbia (Vranjak, Zmajevački potok, and Mileševka Canyon) were investigated by GC and GC/MS analyses. The amount of nonacosan‐10‐ol varied individually from 50.05 to 74.42% (65.74% in average), but the differences between the three investigated populations were not statistically confirmed. The results exhibited variability of the composition of <jats:italic>n</jats:italic>‐alkanes in the epicuticular waxes with their size ranging from C<jats:sub>18</jats:sub> to C<jats:sub>35</jats:sub>. The most abundant <jats:italic>n</jats:italic>‐alkanes were C<jats:sub>29</jats:sub>, C<jats:sub>31</jats:sub>, and C<jats:sub>27</jats:sub> (35.22, 13.77, and 12.28% in average, resp.). The carbon preference index of all the <jats:italic>n</jats:italic>‐alkanes (CPI<jats:sub>total</jats:sub>) of the <jats:italic>P. omorika</jats:italic> populations (average of populations <jats:italic>I</jats:italic>–<jats:italic>III</jats:italic>) ranged from 3.3 to 11.5 (mean of 5.9), while the average chain length (ACL) ranged from 26.6 to 29.2. The principal component and cluster analyses of the contents of nine <jats:italic>n</jats:italic>‐alkanes showed the greatest difference for the population growing in the Mileševka Canyon. The obtained results were compared with previous literature data given for other <jats:italic>Picea</jats:italic> species, and this comparison was briefly discussed.