Background: Knowledge about the modes of transmission and preventive measures of malaria are important preceding factors for the acceptance and use of proven control tools by the community. Objective: To assess knowledge, attitude and practices (KAP) about modes of malaria transmission and preventive methods in the study areas. Methods: A cross-sectional study was conducted in three urban areas of Assosa zone, Western Ethiopia from January to February, 2006. Data analysis was carried out using SPSS for windows version 12.0.1. Adjusted odds ratio and 95% confidence intervals were employed to test the strength of association. Results: About 48% of the study participants were aware that malaria can be transmitted by mosquito bites. Thirty percent (30%), of respondents were aware that mosquitoes carry disease causing microorganism, 95% were aware that mosquitoes bite during night, and 61% were aware that mosquitoes rest at dark places inside the house. Sleeping under a mosquito net and eliminating mosquito-breeding sites were identified by 58% and 52% of respondents, respectively, as major malaria preventive measures. Respondents’ education and wealth status were associated with comprehensive knowledge on malaria preventive measures (OR= 2.42, 95% CI: 1.09, 5.4 and OR= 3.89, 95% CI: 1.99, 7.6, respectively). Conclusion: Knowledge of the role of mosquitoes in malaria transmission and comprehensive knowledge about malaria prevention strategies among the study population were observed to be lower than 50%. Comprehensive behavioral change and communication is required to improve the knowledge of the mode of malaria transmission and its preventive and control measures.