Cutleaf groundcherry (Physalis angulataL.), an annual plant containing a variety of active ingredients, has great medicinal value. However, studies on the genetic diversity and population structure ofP. angulataare limited. In this study, we developed chloroplast microsatellite (cpSSR) markers and applied them to evaluate the genetic diversity and population structure ofP. angulata. A total of 57 cpSSRs were identified from the chloroplast genome ofP. angulata. Among all cpSSR loci, mononucleotide markers were the most abundant (68.24%), followed by tetranucleotide (12.28%), dinucleotide (10.53%), and trinucleotide (8.77%) markers. In total, 30 newly developed cpSSR markers with rich polymorphism and good stability were selected for further genetic diversity and population structure analyses. These cpSSRs amplified a total of 156 alleles, 132 (84.62%) of which were polymorphic. The percentage of polymorphic alleles and the average polymorphic information content (PIC) value of the cpSSRs were 81.29% and 0.830, respectively. Population genetic diversity analysis indicated that the average observed number of alleles (Na), number of effective alleles (He), Nei’s gene diversity (h), and Shannon information indices (I) of 16P. angulatapopulations were 1.3161, 1.1754, 0.1023, and 0.1538, respectively. Moreover, unweighted group arithmetic mean, neighbor-joining, principal coordinate, and STRUCTURE analyses indicated that 203P. angulataindividuals from 16 populations were grouped into four clusters. A molecular variance analysis (AMOVA) illustrated the considerable genetic variation among populations, while the gene flow (Nm) value (0.2324) indicated a low level of gene flow among populations. Our study not only provided a batch of efficient genetic markers for research onP. angulatabut also laid an important foundation for the protection and genetic breeding ofP. angulataresources.