Background and Objectives: Due to the important role of Streptococcus agalactiae, Group B streptococci (GBS), in production of invasive disease in neonates, investigation regarding the pathogenicity and antibiotic resistance factors is necessary in selecting the appropriate therapeutic agents. Beside capsule, the pilus has been currently recognized as an important factor in enhancing the pathogenicity of GBS. Resistance of GBS to selected antibiotics is noticeably increasing which is mainly due to the anomalous use of these drugs for treatment. The aim of this study was to determine the prevalence of pili genes followed by antibiotic susceptibility of GBS, previously serotyped, isolated from pregnant women in the city of Yazd, Iran. Materials and Methods: Fifty seven GBS from pregnant women were subjected to multiplex PCR for determination of PI-1, PI-2a and PI-2b pilus-islands and simultaneously, the phenotype of antibiotic resistance to penicillin, tetracycline, erythromycin, clindamycin, gentamycin and levofloxacin was determined. Antibiotic resistance genes (ermA, ermB, mefA, tetM, int-Tn) were further diagnosed using PCR and multiplex PCR. Results: PI-1+PI-2a with 71.9%; followed by PI-2a (21.1%) and PI-2b (7%) were observed. PI-1+PI-2a in serotype III was (73.2%), serotype II, Ia, Ib and V were 12.2%, 9.8%, 2.4% and 2.4% respectively. GBS penicillin sensitive was 89.5% and 96.5% resistance to tetracycline. The frequency of resistance genes were as follows: tetM (93%), ermA (33.3%), ermB (8.8%), int-Tn (80.7%) and mefA (0). Conclusion: Majority of GBS contained PI-1+PI-2a. Hence presence of this pilus stabilizes the colonization, therefore designing a program for diagnosing and treatment of infected pregnant women seems to be necessary.