The sperm reservoir in the spermatheca of bee queens with desirable genotypes may be depleted. In order to recreate a breeding line, instrumental reinsemination can be performed although beekeepers are generally convinced that ovipositing queens cannot be reinseminated due to the hardening of their valve fold. The beekeeping literature does not provide macroscopic-scale anatomical images of the valve fold. In this two-year study, in total 28 bee queens laying fertilized and unfertilized eggs were subjected to reinsemination with drone semen and ten-fold diluted hematoxylin. The reproductive system was dissected from 24 queens, and the valve fold was subjected to anatomical and histological analysis. The reinseminated queens were aged three months and three years. This study has confirmed that it is possible to carry out instrumental reinsemination in bee queens laying only unfertilized eggs. Afterwards, such queens begin to lay fertilized eggs from which fully developed workers hatch. The mean number of fertilized eggs laid by such queens was 514. The reinsemination of the queens with 2 µl of ten-fold diluted hematoxylin and the histological analyses performed with the use of a stereomicroscope in the macro scale and a light microscope have revealed the actual structure of the stained valve fold, whose shape resembles a “rose flower”. The valve fold consists of one main structure supported by two smaller ones on the sides. In terms of histology, the structure of the valve fold in three-month-old queens is composed of a thick cuticle covering the secretion of epithelial secretory cells and muscles. This structure is responsible for the flexibility of the valve fold. In turn, the valve fold in three-year-old bee queens has an altered histological structure, as its cuticle is lost, leaving only the secretory cells of the epithelium.