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Background and Aim: There is an increasing rate of antibiotic resistance in Neisseria gonorrhoeae (N. gonorrhoeae). Several obstacles affect the culture and susceptibility testing of N. gonorrhoeae. This study was aimed to determine the antimicrobial susceptibility pattern and molecular characteristics in multidrug-resistant (MDR) N. gonorrhoeae in Jakarta, Indonesia. Materials and Methods: Forty-one endocervical specimens were streaked onto the chocolate agar and then gently rolled onto a glass object. The suspected colonies were identified by Gram staining, oxidase testing, and biochemical identification using VITEK® 2 NH cards. The antimicrobial susceptibility testing was performed using the disk diffusion or E-test method. Molecular detection of N. gonorrhoeae was carried out using the SYBR green real-time PCR targeting the opa gene. The multiplex real-time PCR with high-resolution melting (HRM) was also conducted to analyze the gene mutations in penA and 23S rRNA. DNA sequencing was used to confirm penA and 23S rRNA mutations. Results: SYBR green real-time PCR against the opa gene on direct specimens detected N. gonorrhoeae with higher positivity rate (49%) compared to the culture (34%). Susceptibility testing detected 11.1% N. gonorrhoeae resistant to cefixime, whereas resistance to azithromycin and ceftriaxone was 0%. The resistance of N. gonorrhoeae to levofloxacin and kanamycin was 33.3% and 88.9%, respectively. All isolates were resistant to penicillin, tetracycline and ciprofloxacin. There were no mutations in the penA genes Ala501, Gly545Ser, 23S rRNA A2059G and C2611T. Conclusion: Cefixime resistance was found in our samples. This resistence was not correlated with penA gene mutations. All isolates were still susceptiblle to azithromycin, which was in line with its molecular characteristics. The SYBR green real-time PCR targeting the opa gene successfully detected the DNA extracted from endocervical swabs as N. gonorrhoeae. [ABSTRACT FROM AUTHOR]