The study aimed to determine the reasons for polio vaccine hesitancy among parents of persistently missed children (PMCs) in the high-risk areas of Karachi, Pakistan. A cross-sectional survey of parents of PMCs was conducted in April 2019 in 34 high-risk union councils of Karachi. PMCs were randomly selected from the polio program database, and further information was collected on a questionnaire by trained staff using face-to-face interviews with parents of PMCs. A total of 325 participants were included in the study. Among refusals, 112 (37.3%) had no trust in vaccine quality, followed by 45 (15.0%) who were afraid of side effects, 42 (14.0%) whose elders did not allow polio vaccination, 39 (13.0%) who refused due to the influence of negative social media videos, and 20 (6.7%) who had no trust in polio teams. We concluded that misconception is still a big challenge, and the program needs to strive for community acceptance. Low levels of trust in vaccines and teams as well as fear of OPV side effects were among the main reason for vaccine hesitancy. The participant communities recommended involving famous medical doctors, religious influencers, and TV or sports stars to enhance knowledge and acceptance of polio vaccination.