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In South America, knowledge of major spawning sites is crucial for maintaining migratory fish populations. In this study, we aimed to understand the spatio-temporal distribution of fish eggs in the upper São Francisco River using high sampling frequency and DNA metabarcoding identification. We evaluated the possible effects of the non-molecular identification of eggs and decreased sampling frequency on the determination of spawning sites and major breeding periods. Collections were carried out every three days from November 2019 to February 2020. We found that, if we had assumed that all of the free and non-adhesive sampled eggs belonged to migratory species, as is usual in the literature, this assumption would have been wrong for both the spawning sites and the breeding periods. Moreover, any decrease in the frequency of sampling could dramatically affect the determination of the major spawning rivers, and the spawning events of some of the migratory species may not have been detected. Therefore, without the proper identification and adequate sampling frequency of eggs, important spawning sites may be overlooked, leading to ineffective or inappropriate conservation measures.