Abstract: We report new sequence types of 14 penicillinase-producing Neisseria gonorrhoeae, isolated from sexually transmitted disease clinic attendees in Manaus, Brazil. They were characterized by WI/WII/WIII groups, susceptibility testing and Multi-Antigen Sequencing Typing/Mutilocus Sequence Typing protocols. Twelve were classified as WII/III and 2 as WI and were presented resistance to penicillin and tetracycline. New alleles for por and AroE genes and novel sequence types were identified, revealing molecular characteristics not described previously. ST1590 is the common ancestor after eBURST analysis, and these findings represent an important contribution of molecular epidemiology approach in gonococci’s research in Amazonas.