Mustelidae is the largest and most diverse family in the order Carnivora. The phylogenetic relationships among the subfamilies have especially long been a focus of study. Herein we are among the first to employ two new introns (4 and 7) of the nuclear β-fibrinogen gene to clarify these enigmatic problems. In addition, two previously available nuclear (IRBP exon 1 and TTR intron 1) and one mt (ND2) data sets were also combined and analyzed simultaneously with the newly obtained sequence data in this study. Detailed characterizations of the two intronic regions not only reveal the remarkable occurrences of short interspersed element (SINE) insertion events, providing a new example supporting the attractive hypothesis that attrition of an earlier retroposition may offer a proper environment for successive retropositions by forming a “dimer-like” structure, but also demonstrate their utility in the resolution of mustelid phylogeny. All of our analyses confirm the assemblage of Mustelinae, Lutrinae, and Melinae with confidence; moreover, two clades within Mustelinae were clearly recognized, i.e., genera Mustela and Martes. Notably, genus Martes of Mustelinae was found to branch off first, followed by Melinae and then a clade containing Lutrinae and genus Mustela of Mustelinae, indicating paraphyly of Mustelinae. In addition, Mephitinae diverges before the other mustelids and the monophyletic Procyonidae in all cases, supporting its elevation to a separate family. Additional independent genetic markers are still in need to resolve the trichotomy among Mephitinae and the other two carnivoran clades, Ailuridae and Procyonidae/non-mephitine Mustelidae.