Five rabbit populations of New Zealand White (NZW), Californian (CAL), crossbred NZW×GW and two generations of the synthetic population – SPF₁ and SPF₂ reared in Bulgaria were included in the present study with the aim of detecting the genetic variability of the growth hormone encoding gene (<em>GH</em>) via polymerase chain reaction with the restriction fragment length polymorphism analysis and direct sequencing. The targeted region of the rabbit <em>GH</em> gene was amplified and a fragment of a total of 231 bp was obtained in all studied populations. Allele identification was determined after enzymatic digestion, where two fragments of 62 and 169 bp correspond to allele C and an undigested fragment of 231 bp corresponds to allele T. Two additional bands of 107 and 124 bp evidenced A/G genetic polymorphism in the rabbit <em>GH</em> gene. Thirtyeight percent of the studied rabbits were carriers of the double mutation (C/T+A/G) in the same locus as the studied <em>GH</em> gene. The sequence analysis revealed two nucleotide substitutions – g.111C&gt;T and g.156A&gt;G in the non-coding region between the regulatory TATA box and 5’ UTR region, and a novel g.255G&gt;A genetic variant in intron 1 of GH gene. The A&gt;G transition was most frequent (40.57%), compared to the other ones, G&gt;A (28.57%) and C&gt;T (10.80%), respectively. The most frequent genotype in the NZW population was homozygous TT (0.93), with a prevalence of the T allele (0.97) over allele C (0.03) for g.111C&gt;T SNP site. The distribution of the allele and genotype frequencies at the sites g.156A&gt;G and g.255G&gt;A in this rabbit group was identical, with the highest value of 0.93 for alleles A and G, respectively. The rabbit populations CAL and NZW×GW showed equal frequencies of the prevalent T allele (0.83) and for homozygous TT genotype (0.67) according to g.111C&gt;T SNP. The highest values were obtained for the allele А (0.83) and for homozygous AA genotype (0.67) at c.33A&gt;G SNP in these rabbit groups. The highest values (0.67, 0.60 and 0.80) for the heterozygous genotypes at g.111C&gt;T, g.156A&gt;G and g.255G&gt;A SNPs, respectively, were detected among the SPF₂ rabbit population, compared to the both homozygous genotypes. The results obtained in the present research indicates a significant degree of genetic variability of the studied polymorphic <em>GH</em> locus in the SPF₂ rabbit group.