This study investigates (a) whether there is a functional neural activation at the frontal and temporal brain regions during the comprehension of Malay relative clause (RC), and (b) the differences in the activated areas among native (L1) and non-native (L2) Malay language speakers. The subject relative clause (SRC), object relative clause (ORC), and subject-verb-object (SVO) were used as the study stimuli. Participants were asked to do a sentence-picture matching task during an fMRI measurement. The random-effect analysis (RFX) using two-way ANOVA was conducted for the fMRI data. The main effect of the group at the puncorrected < 0.001, cluster size > 20 voxels found that the comprehension of Malay relative clauses had activated frontal and temporal brain regions in L1 and L2. The multiple comparisons of L1>L2 showed a significant difference left-lateralised in the temporo-parietal region. While for L2>L1, the significant activations were indicated as distributed to the frontal, temporal, parietal, and occipital regions that lateralised to the right hemisphere. Conclusions: The findings suggested that the comprehension of Malay relative clauses had caused the activation at different brain regions amongst L1 and L2 groups. It was also found that both L1 and L2 groups showed their preference in SRC, the mean reaction time showed that they had a faster reaction time to comprehend SRC than ORC. The findings from this study can also be applied in clinical language intervention, and it is expected to benefit children and adults with speech and language disorders.