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The study aimed to analyze the genetic diversity in the Czech population of <i>Apis mellifera</i> using mitochondrial DNA markers, <i>tRNA^leu-cox2</i> intergenic region and <i>cox1</i> gene. A total of 308 samples of bees were collected from the entire Czech Republic (from colonies and flowers in 13 different regions). Following sequencing, several polymorphisms and haplotypes were identified. Analysis of <i>tRNA^leu-cox2</i> sequences revealed three <i>Dra</i>I haplotypes (C, A1, and A4). The <i>tRNA^leu-cox2</i> region yielded 10 C lineage haplotypes, one of which is a newly described variant. Three A lineage haplotypes were identified, two of which were novel. A similar analysis of <i>cox1</i> sequences yielded 16 distinct haplotypes (7 new) within the population. The most prevalent <i>tRNA^leu-cox2</i> haplotype identified was C1a, followed by C2a, C2c, C2l, and C2d. For the <i>cox1</i> locus, the most frequent haplotypes were HpB02, HpB01, HpB03, and HpB04. The haplotype and nucleotide diversity indices were high in both loci, in <i>tRNA^leu-cox2</i> with values of 0.682 and 0.00172, respectively, and in <i>cox1</i> 0.789 and 0.00203, respectively. The Tajima’s D values were negative and lower in <i>tRNA^leu-cox2</i> than in <i>cox1</i>. The most frequent haplotypes were uniformly distributed across all regions of the Czech Republic. No haplotype of the indigenous M lineage was identified. High diversity and the occurrence of rare haplotypes indicate population expansion and continuous import of tribal material of the C lineage.