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Abstract Background COVID-19 mass vaccination is the only hopeful savior to curb the pandemic. Vaccine distribution to achieve herd immunity is hindered by hesitance and negative attitude of the public against COVID-19 vaccination. This study aims to evaluate the vaccine hesitancy and attitudes in major cities in Pakistan as well as their determinants. Methods A cross-sectional telephonic survey was conducted in June 2021 in major cities of Pakistan including Karachi, Lahore, Islamabad, Peshawar, and Gilgit, from unvaccinated urban population aged 18 years or older. Random Digit Dialing through multi-stage stratified random sampling was used to ensure representation of each target city and socio-economic classes. Questionnaire collected information on socio-demographics, COVID-19-related experiences, risk perception of infection, and receptivity of COVID-19 vaccination. Multivariate logistic regression analyses were performed to identify key determinants of vaccine hesitancy and acceptance. Results The prevalence of vaccinated population in this survey was 15%. Of the 2270 respondents, 65% respondents were willing to vaccinate, while only 19% were registered for vaccination. Factors significantly associated with vaccine willingness were older age (aOR: 6.48, 95% CI: 1.94–21.58), tertiary education (aOR: 2.02, 95% CI: 1.36, 3.01), being employed (aOR: 1.34, 95% CI: 1.01, 1.78), perceived risk of COVID-19 (aOR: 4.38, 95% CI: 2.70, 7.12), and higher compliance with standard operating procedures (aOR: 1.72, 95% CI: 1.26, 2.35). The most common vaccine hesitancy reasons were ‘no need’ (n = 284, 36%) and concerns with ‘vaccine safety and side effects’ (n = 251, 31%), while most reported vaccine motivation reasons were ‘health safety’ (n = 1029, 70%) and ‘to end the pandemic’ (n = 357, 24%). Conclusions Although our study found 35% hesitancy rate of COVID-19 vaccine, there were noticeable demographic differences that suggest tailored communication strategy to address concerns held by most hesitant subpopulation. Use of mobile vaccination facilities particularly for less mobile and disadvantaged, and implementation and evaluation of social mobilization strategy should be considered to increase overall COVID-19 vaccination acceptance and coverage.