Functional MRI (fMRI) is gaining importance in the preoperative assessment of language. Selecting the appropriate language to test by fMRI in trilingual patients is intricate. Our objective is to compare fMRI maps for all three languages in left- and right-handed trilingual subjects. 15 right- and 15 left-handed trilingual volunteers were included in the study. We performed fMRI for each volunteer with a visual responsive naming paradigm that was repeated three times, once in each language. The activated areas and the laterality indices were calculated and correlation with the age of acquisition and proficiency of each language was determined. Strong statistical correlation was found between the Laterality Index (LI) of the three languages, in both the right and left-handed groups. Discordant lateralization of language was only observed in four left-handed subjects who demonstrated bilateral and left-lateralization. In right-handed subjects, the activation maps for the first and the second acquired language were similar. The largest activation was seen with the last acquired language. Irrespective of language proficiency and age of acquisition, the language lateralization might change for left-handed subjects. In right-handed subjects, there is no change and the last acquired language results in the largest activation. fMRI performed for a single language can accurately determine language lateralization in right-handed subjects, whereas in left-handed subjects, it is mandatory to test all languages.