Abstract: Education has a vital role in development of any country in the world. Part of it is teaching and learning process which involving two very basic actors, teachers and students. It has been admitted that the success or fail of human development determined by their personality. This is a trait which will influence human behavior. One of the important aspects of human personality, according to some of Organizational Behavior text books, is called locus of control (LOC) which its measurement initiated by Rotter (1978), but for Indonesian students still need to be validated. Theoretically, it determines knowledge, then indirectly affect on behavior (Blaikie, 1993). How to develop and validating LOC instrument and its relation to student’s knowledge about ecosystem based on students gender is the objective of this study. Therefore, a survey method has been conducted in three different big cities, Palembang, Jakarta, and Makassar which involving senior high school students with total n = 362. Some LOC indicators developed based on its conceptual definitions, with two-forced choice item (17 items) by 2-1 scores. Student’s knowledge about ecosystem instrument consists of 17 items as well with "true-false choices.” Research results reveal that there is no significant difference between male and female students LOC and knowledge about ecosystem. Only one item of LOC is not valid and all knowledge items are valid. Reliability of LOC instrument is .705, but low reliability found on knowledge, .402. These findings come up with conclusion that students LOC could be validated empirically and students LOC could be used to predict students’ knowledge about ecosystem, both for male and female students which supposed to, finally, determine students responsible behavior in facing environmental issues.