Background: Cervical cancer is one of the most common cancers in the female reproductive system. Awareness of cervical cancer diagnosis, risk factors and screening can lead to rapid treatment and their well-being. Methods: This cross-sectional study was conducted on 1900 female health workers in Isfahan province and 230 similar women in Chaharmahal and Bakhtiari province. A researcher-made questionnaire was used to collect data about knowledge and attitude. Numerical outcomes were analyzed using ANOVA and linear regression methods and categorical outcomes were applied to the chisquared test. Data were analyzed at 5% significant level using SPSS 23. Results: The result of linear regression showed that there was a significant difference in the mean score of knowledge about Pap smear in both provinces (P = 0.001). There was a significant relationship between the mean scores of knowledge about Pap smear in Isfahan province and marriage at an early age (P = 0.001), smoking (P = 0.005) and Chaharmahal and Bakhtiari province with cervical ulcer (P = 0.03). In addition, there was a significant difference in the mean score of attitude toward Pap smear between both provinces (P = 0.001) and there was a significant relationship between the mean scores attitude toward Pap smear in Isfahan province and marriage at an early age (P = 0.001), cervical ulcer (P = 0.002), and smoking (P = 0.001) and Chaharmahal and Bakhtiari province with genital infection (P = 0.012) increase the number of pregnancies (P = 0.002). Conclusions: Considering the importance of knowledge and attitude of female health worker in both provinces, it is necessary to carry out educational programs especially in the provinces of Chaharmahal and Bakhtiari.