Previous research has demonstrated an association between tonality and mood as well as an association between timbre and mood. However, there has been lots of inconsistency in the findings from previous research, which could be due to the differences in stimuli creation, recording, and presentation. In our study, we used isolated arpeggios created on MIDI software as stimuli to investigate how instrument timbre playing musical chord affects people’s tonality perception when the tonality of the chord is ambiguous, without explicitly mentioning mood except measured at the end of the study. 49 UChicago-affiliated adults listened to arpeggios played on 5 different instruments and with 5 different tuning steps of the middle note and made judgments on whether each chord was major or minor. Results showed that timbre and tuning step both significantly influenced listeners’ tonality perception, and there was a significant interaction between timbre and tuning. There was also a significant difference in perceived mood associated with each instrumental timbre, which followed a similar trend to that for the proportion of major tonality judgment. Arpeggios played on timbres that were rated as “happier” were more likely to be categorized as major, while those played on timbres that were rated as “sadder” were more likely to be categorized as minor. Arpeggios played on oboe had the smallest proportion of major judgments, while those played on xylophone had the largest. Findings from this study built a foundation for further research in the timbre-mood-tonality connection. Future research could investigate whether the same results hold true for artificial timbre and for stimuli other than three- note arpeggios.