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A cross-sectional study was conducted to determine the vaccination status and barriers to vaccination among the university students by utilizing a simple random sampling technique in the largest public sector university of Southern Punjab, Pakistan. The participants comprised 380 university students. Data was collected by a self-designed questionnaire. Statistical Package for Social Sciences (SPSS) was used for data analysis. Chi-square Test and Fischer Exact test were applied to assess the impact of demographics on vaccination status, and barriers to vaccination. Out of 380 participants, 328 (86.31%) were males and 52 (13.68) females. The immunization status of university students against various diseases was variable: 97.10% (n=369) were vaccinated against poliomyelitis, 58.68% (n=223) against BCG, 44.21% (n=168) against hepatitis B, 49.21% (n=187) against diphtheria, pertussis and tetanus and 55.26% (n=210) against measles vaccine. The barriers to vaccination were unwillingness 15.0% (n=57), inaccessibility 17.10% (n=65), financial issues 4.47 % (n=17) and unawareness 63.42% (n=241). Moreover, 31% (n=118) of the participants considered that the use of vaccines is unsafe. The vaccination status of the university students in Southern Punjab, Pakistan is alarming as most of the students were unvaccinated. The unawareness and perception of the unsafety of vaccines were the biggest barriers to vaccination.