Salient sensory experiences often have a strong emotional tone, but the neuropsychological relations between perceptual characteristics of sensory objects and the affective information they convey remain poorly defined. Here we addressed the relationship between sound identity and emotional information using music. In two experiments, we investigated whether perception of emotions is influenced by altering the musical instrument on which the music is played, independently of other musical features. In the first experiment, 40 novel melodies each representing one of four emotions (happiness, sadness, fear, or anger) were each recorded on four different instruments (an electronic synthesizer, a piano, a violin, and a trumpet), controlling for melody, tempo, and loudness between instruments. Healthy participants (23 young adults aged 18-30 years, 24 older adults aged 58-75 years) were asked to select which emotion they thought each musical stimulus represented in a four-alternative forced-choice task. Using a generalized linear mixed model we found a significant interaction between instrument and emotion judgement with a similar pattern in young and older adults (p < .0001 for each age group). The effect was not attributable to musical expertise. In the second experiment using the same melodies and experimental design, the interaction between timbre and perceived emotion was replicated (p < .05) in another group of young adults for novel synthetic timbres designed to incorporate timbral cues to particular emotions. Our findings show that timbre (instrument identity) independently affects the perception of emotions in music after controlling for other acoustic, cognitive, and performance factors.