MicroRNA (miRNA) 5'-isoforms, or 5'-isomiRs, are small-RNA species that originate from the same genomic loci as the major miRNAs with their 5' ends shifted from the 5' ends of the miRNAs by a few nucleotides. Although 5'-isomiRs have been reported, their origins, properties and potential functions remain to be examined. We systematically studied 5'-isomiRs in human, mouse, fruitfly and worm by analysing a large collection of small non-coding RNA and mRNA profiling data. The results revealed a broad existence of 5'-isomiRs in the four species, many of which were conserved and could arise from genomic loci of canonical and non-canonical miRNAs. The well-conserved 5'-isomiRs have several features, including a preference of the 3p over the 5p arms of hairpins of conserved mammalian miRNAs, altered 5'-isomiRs across species and across tissues, and association with structural variations of miRNA hairpins. Importantly, 5'-isomiRs and their major miRNAs may have different mRNA targets and thus potentially play distinct roles of gene regulation, as shown by an integrative analysis combining miRNA and mRNA profiling data from psoriatic and normal human skin and from murine miRNA knockout assays. Indeed, 18 5'-isomiRs had aberrant expression in psoriatic human skin, suggesting their potential function in psoriasis pathogenesis. The results of the current study deepened our understanding of the diversity and conservation of miRNAs, their plasticity in gene regulation and potential broad function in complex diseases.