We report the development of the Assessment of Individual Motives-Questionnaire (AIM-Q), a new instrument based on an evolutionary psychology theory of human motivation. It provides multitrait-multimethod (MTMM) assessment of individual differences on 15 motive scales. A total heterogeneous sample of N = 1,251 participated in eight studies that evaluated the homogeneity, internal consistency, test-retest reliability, and MTMM convergent and discriminant validities of the AIM-Q's three methods. These studies generally support the overall strategy of assessing individual differences in multiple evolutionary-based motives with multiple methods. Additional validity studies are underway and, when validated further, the AIM-Q may offer a promising option for evolutionary psychologists and behavioral geneticists wanting to incorporate individual differences into their research but have had to use existing self-report measures of personality, which were not designed for such a purpose. It may also offer clinical and counseling psychologists an additional approach to personality measures for the prediction of behavior. (Contains 12 tables.)