Background: In an era of precipitous insect declines, effective entomology education is especially needed to support firsthand knowledge of nature. Understanding what students know and feel about insects is instrumental to teaching and curriculum development. This study describes the development and validation of a new survey instrument, EntoEdu, measuring 'entomology literacy', based on attitude and knowledge, in a cross-cultural context. For the survey validation we use data collected from students in Czechia (CZ), a country known for its entomophilia, and the United States of America (US) to demonstrate the utility of this survey and to address the questions: how do entomology attitude and knowledge differ across national affiliation and study domain, and how are entomology attitude and knowledge correlated in the context of these differences? Results: Based on responses from 635 first-year college students, we demonstrate high reliability and evidence of validity of the EntoEdu instrument. Factor analysis supports five independent attitudinal categories within the instrument: "Intention to Engage with Insects," "Attitude toward Behavior," "Control Belief," "Hobby," and "Disgust." In this study population, average attitude scores did not differ with nationality, whereas knowledge scores were higher in CZ than in the US. In both countries, attitude and knowledge scores were higher among biology students than those in other study domains, and attitude and knowledge were positively correlated. Conclusions: The EntoEdu instrument, based on globally recognizable insect taxa, ecology, and behavior, has been developed for broad utility in assessing attitudes toward and knowledge of insects at the post-secondary level, with potential for use at both lower (K-12) and higher (advanced university) levels. The instrument is presented here in two language versions and can be translated into additional languages for comparison of results across test populations in additional countries. In our initial test population we find attitude and knowledge to be correlated, both of which are influenced by nationality, with Czechs more knowledgeable about insects than their US counterparts. We anticipate that this instrument will facilitate entomology assessment to help tailor biology education programs to students' actual, rather than assumed, entomology knowledge and attitudes, and for tracking change over time.