Plants in the Vallisneria genus have similar external morphologies, with linear or strap-shaped leaves, making it difficult to directly identify species in the field. But the number of stamens in the male flowers is one of the distinguishing characteristics of species in Vallisneria Linn. The male flowers of Vallisneria species are small, measuring only 1.5 mm. Identifying the number of stamens in these male flowers through manual dissection and observation methods can be challenging. This study utilized a hydroponic pre-culture method with male plants and male flower primordia of Vallisneria to obtain naturally opened male flowers. Anatomical observations and research on the male flowers, fruits, and seeds of three Vallisneria species were conducted using anatomical microscopes.Microscopic observations revealed that in V. natans, there is one stamen in the male flowers. The fruits are cylindrical in shape, with a smooth surface, and the seeds are wingless but have longitudinal stripes. In V. spinulosa, there are two stamens in the male flowers. The fruits are triangular in shape, with spines along the edges, and the seeds have wings. In V. denseserrulata, there are also two stamens in the male flowers. The fruits are cylindrical with three edges, have a smooth surface, and the seeds are wingless.Based on literature and introduction cultivation experiments, V. denseserrulata, V. spinulosa, V. longipedunculata and V. anhuiensis are reported as new distribution records of Vallisneria Linn in Anhui Province. Among them, V. longipedunculata and V. anhuiensis are new species found in Anhui.