As a class of small non-coding RNAs, the negative regulatory RNAs have been widely studied, but little focus has been placed on the loop sequences. Indeed, the loops, including the loop structure and sequence, may tune and alter miRNA activity, which may further affect miRNA expression by affecting Dicer recognition and cleavage during miRNA maturation. The potential roles of loop sequences during miRNA biogenesis have been studied, especially for occurrence of multiple isomiRs. These isomiRs are mainly derived from imprecise and alternative cleavage by Drosha and Dicer during miRNA processing and maturation. These loop sequences may provide more information for miRNA biogenesis, especially based on the analysis of loop sequences across different animal species. In the present study, let-7 gene family loop sequences and other relevant miRNAs were analyzed and clustered to elucidate evolutionary and functional roles relating to miRNA biogenesis. Multicopy miRNA genes may be located on different chromosomes and are mainly derived from historical duplication events. Although the same mature miRNAs can be derived from these multicopy pre-miRNAs, the arms and loop sequences can be involved in larger divergence events, particularly the loops. These loops may show diverse genetic distances relative to other homologous miRNA genes, and are always grouped in different clusters. Interestingly, we found changed Ginsenoside-Ro length distributions of the loop sequences across different animal Estradiol Benzoate species and among different homologous miRNAs. In higher metazoan species, the let-7 loops tend to be longer than those seen in lower species, with varying length distributions also seen different homologous miRNAs. Clustered miRNAs tend to have similar length distribution, which implicates that the loop lengths may be affected by evolutionary relationships. Additionally, longer loop sequences may be an evolutionary trend in let-7 gene family, which may be of importance during miRNA biogenesis. Specifically, loop length may influence the stem-loop structure and stability, with longer loop sequences providing a possibility to dominate the evolution of miRNA genes across different animal species and homologous miRNAs within a specific species. These length variances further increase loop sequence diversity, which contributes to the evolutionary divergence between different miRNA genes, including homologous miRNAs. Indeed, although varied loop sequences exist, we still found the potential nucleotide characteristics in the 59 and 39 ends of the loops. Dominant nucleotides, such as uracil and guanine, are present at higher frequencies at the terminal ends, with these biased nucleotide compositions possibly influencing Dicer cleavage and contributing to the phenomenon of multiple isomiRs. While many isomiRs can be produced from a miRNA loci, only several isomiRs are dominantly expressed. Some let-7 sequences are located in gene clusters with homologous miRNAs or other miRNAs, such as with the mir125 and mir-99 gene families. Loops of clustered miRNA gene families may have different lengths, suggesting the length difference of loops from different miRNA genes. Significant differences in 59 and 39terminal nucleotide compositions are noted among these clustered miRNAs, with both uracil and guanine dominating in the two terminus ends. Compared to the stable length distributions of miRNAs, rapid loop sequence evolution can drive miRNA gene evolution and may further affect isomiR expression profiles during pre-miRNA processing. Expression analysis indicates stable isomiR expression profiles, even across different tissues and animal species, suggesting stable Drosha and Dicer cleavage during miRNA processing and maturation.