| Summary: | <i>Coxiella burnetii</i>, the zoonotic agent of Q fever, has a worldwide distribution including Iran. However, no information regarding the circulating genotype of this infection has been reported in Iran. This study aimed to evaluate the genetic diversity of <i>C. burnetii</i> in Iran using the multi-spacer sequence typing (MST) method. First, 14 positive <i>C. burnetii</i> samples (collected from four sheep, three goats, and seven cattle) were confirmed using quantitative polymerase chain reaction (qPCR) targeting the <i>IS1111</i> gene. Then, ten spacers (Cox 2, 5, 18, 20, 22, 37, 51, 56, 57, and 61) were amplified using PCR for future MST analysis. The <i>in-silico</i> MST genotyping analysis of domestic ruminant samples revealed two new alleles (Cox5.11 and Cox56.15) in Cox5 and Cox56 loci that led to the emergence of four novel MST genotypes (MST62, 63, 64, and 65) and one MST genotype that has been previously described (MST61). This study showed the circulation of five MST <i>C. burnetii</i> genotypes among Iranian domestic ruminants. Understanding the <i>C. burnetii</i> genotypic profiles is critical in determining and preventing Q fever outbreaks.
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