Evaluation of genetic variation was performed within 62 local maize populations originating from Slovakia and Czech Republic. In total 48 alleles at 22 analyzed isoenzyme loci with an average of 2.2 alleles per locus were revealed. The percentage of polymorphic loci ranged from 14% to 59% and the frequencies of detected alleles varied from null to four per locus. No polymorphism was detected at the loci <i>Dia2</i>, <i>Got3</i>, <i>Mdh4</i>, <i>Mmm</i>, and <i>Pgm1</i>. The highest number of alleles (four) was detected at loci <i>Acp1</i>, <i>Cat3</i>, <i>Pgm2</i>. No new alleles were identified, nevertheless the frequency of seven alleles was only about 1%. The expected heterozygosity ranged from null to 0.492 with an average of 0.197. The revealed isoenzyme polymorphism confirmed that all analyzed populations were heterogeneous and as many as 17 of them were completely heterogeneous. None of the analyzed populations was identical in the frequency of alleles at all 22 analyzed loci.