The objectives of this study were to determine differences in knowledge and perception of animal welfare (with emphasis on dairy cattle) among undergraduate students from two universities with different missions and visions and between students from different faculties. One thousand surveys were obtained from Universidad de Chile (UChile; n = 500) and Pontificia Universidad Católica de Chile (PUC; n = 500) students. The students from both universities were from the following faculties: Agronomy, Architecture, Biology, Economic Sciences, Psychology, Law, Philosophy, Basic Education, Civil Engineering and Medicine. The majority (77%) of students from both universities were aware of animal welfare. Most (56%) students understand animal welfare as the ’mental and physical state of animals’. Regardless of their faculty, around 97% of the total respondents perceived animal welfare as important for production systems. Regarding specific knowledge about cows’ welfare related to milk management and behavior, students from Economic Sciences, Psychology, Law, Philosophy, Basic Education, and Civil Engineering had less (p < 0.001) self-reported knowledge about cows´ basic behavior and specific management practices such as milking and were more negative in their perceptions of dairy production. Overall, results showed that the students’ faculty explained most of the differences among undergraduate students in relation to their perceptions and knowledge about animal welfare. Our data is important, as undergraduate students will make purchasing and power decisions as well as having potential influence on future policies that could modify the animal production industry.