Background: Puerariae Lobatae Radix is widely used in the pharmaceutical, food, and cosmetic industries. For these applications, when grading roots according to diameter, thick roots are always considered to be of better quality. Objective: The objective of this study is to qualitative and quantitative analysis of isoflavonoids profiling in various tissues of P. lobata roots. Materials and Methods: Ultraperformance liquid chromatography quadrupole/time-of-flight-mass spectrometry (UPLC-QTOF/MS) and high-performance liquid chromatography separation and ultraviolet-visible detection were used to identify and profile the isoflavonoids detected in various tissues of P. lobata roots. Results: Consequently, 82 peaks were detected by UPLC-QTOF/MS, and 28 isoflavonoid compounds were identified in the various tissue samples. According to the results of this study, the thick roots of P. lobata are of better quality for medical use than the thin ones. The fine roots and the periderm of roots contained slightly lower isoflavonoid abundances. The results indicated that isoflavonoids, particularly puerarin, mainly accumulated in the xylem and phloem. Conclusions: This study provides a new and practical method for evaluating the quality of P. lobatae Radix.