MicroRNAs (miRNAs) have been proven to play important roles at the post-transcriptional level in animals and plants. To investigate clustering patterns and specific sequence characteristics in the flanking regions of plant miRNA genes, we performed genome-wide analyses of Arabidopsis thaliana, Populus trichocarpa, Oryza sativa and Sorghum bicolor. Our results showed that miRNA pair distances were significantly higher than would have been expected to occur at random and that the number of miRNA gene pairs separated by very short distances of < 1 kb was higher than of protein-coding gene pairs. Analysis of the promoter architecture of different miRNA genes in plants revealed significant differences in the number and distribution of core promoters between intergenic miRNAs and intragenic miRNAs, and between highly conserved miRNAs and low conserved or nonconserved miRNAs. We applied two motif-finding algorithms to search for over-represented, statistically significant sequence motifs, and discovered six species-specific motifs across the four plant species studied. Moreover, we also identified, for the first time, several significantly over-represented motifs that were associated with conserved miRNAs, and these motifs may be useful for understanding the mechanism of origin of new plant miRNAs. The results presented provide a new insight into the transcriptional regulation and processing of plant miRNAs.