Background:  Staphylococcus is a Gram-positive (GM⁺) facultative anaerobic, non-motile coccus. It's frequently results in a variety of illnesses in human. Staphylococcus sp. considered the most common organisms that can infect the thyroid gland. Methods: Genomic DNA was extracted from 20 thyroiditis patients' blood samples and analyzed for 16S rRNA using PCR, employing the Universal gene. Gene amplification was performed in a thermocycler under specific cycling conditions, including initial denaturation, annealing, extension, and final extension. For the purpose of partially DNA sequencing for the 16S rRNA gene, six PCR results were selected in order to investigate any possible association between the results and the global records in GenBank. The data was analyzed using Statistical Package for Social Sciences (SPSS) version 27, using chi-square test and basic ratios, with P-values between 0.05 and 0.01 indicating statistical significance. Results: Findings showed that 6/20 (30%) were Staphylococcus sp. isolates. The 16S rRNA PCR product was registered in GenBank in accordance with the official accession numbers of (PP396084, PP396083, PP396085, PP396086, PP396082 and PP396093). The local Staphylococcus sp. isolates have distinct molecular links with those of similar strains worldwide according to the phylogenetic tree generated by the MEGA-10 program. Conclusions: Thyroiditis continues to be one the most threating health problems around the world. Thyroiditis patients are more susceptible to GM⁺ microorganisms. Staphylococcus sp. is an important opportunistic pathogen and it's associated with substantial morbidity rate.