Background: Streptococcus agalactiae or Group B Streptococcus is an important cause of maternal complications in pregnancy and neonatal infections leading to potentially severe ailments due to transmission during delivery. This study aimed to detect Streptococcus agalactiae in pregnant women by culture and molecular method for early detection and prevention of infection. Methods: A total of 90 vaginal swab samples were collected from Index Medical College Hospital & Research Centre, Indore and were subjected to culture, Gram staining, biochemical reactions, and molecular method i.e., Polymerase Chain Reaction. Result: 5 out of 90 (5.6%) samples showed growth of Group B Streptococcus by culture. PCR detected GBS in 24 samples. This yielded a positivity rate of 26.67%. All the positive isolates were subjected to antimicrobial susceptibility testing by disc diffusion. It was found that all isolates were susceptible to Penicillin (100%) and Vancomycin (100%). Maximum resistance was seen with Tetracycline (25%) followed by Erythromycin (20.83%), Linezolid (12.50%) and Clindamycin (8.33%). Interpretation: Similar rates of colonization are seen in different parts of the country when culture is the primary method of detection whereas, using a molecular method significantly increases the rates of detection Conclusion: Timely detection of GBS is pivotal in preventing neonatal morbidity and eventually, mortality owing to neonatal meningitis. Antenatal screening for GBS at 35-37 weeks of gestation is recommended. Although culture is the gold standard method for detection, the colonization rates may be under-recognized or under-documented if molecular method like PCR is not used. Penicillin allergy can lead to serious anaphylaxis. Hence, it is important to know the antimicrobial susceptibility pattern of the pregnant women.