Abstract Background MicroRNAs (miRNAs) repress target genes at the post-transcriptional level, and function in the development and cell-lineage pathways of host species. Tissue-specific expression of miRNAs is highly relevant to their physiological roles in the corresponding tissues. However, to date, few miRNAs have been spatially identified in the silkworm. Results We establish for the first time the spatial expression patterns of nearly 100 miRNAs in multiple normal tissues (organs) of Bombyx mori females and males using microarray and Northern-blotting analyses. In all, only 10 miRNAs were universally distributed (including bmo-let-7 and bmo-bantam), while the majority were expressed exclusively or preferentially in specific tissue types (e.g., bmo-miR-275 and bmo-miR-1).... Additionally, we examined the developmental patterns of miRNA expression during metamorphosis of the body wall, silk glands, midgut and fat body. In total, 63 miRNAs displayed significant alterations in abundance in at least 1 tissue during the developmental transition from larvae to pupae (e.g., bmo-miR-263b and bmo-miR-124). Expression patterns of five miRNAs were significantly increased during metamorphosis in all four tissues (e.g., bmo-miR-275 and bmo-miR-305), and two miRNA pairs, bmo-miR-10b-3p/5p and bmo-miR-281-3p/5p, showed coordinate expression. Conclusions In this study, we conducted preliminary spatial measurements of several miRNAs in the silkworm. Periods of rapid morphological change were associated with alterations in miRNA expression patterns in the body wall, silk glands, midgut and fat body during metamorphosis. Accordingly, we propose that corresponding ubiquitous or tissue-specific expression of miRNAs supports their critical roles in tissue specification. These results should facilitate future functional analyses.