||Genomics-based, longitudinal comparisons between ex situ and in situ agrobiodiversity conservation strategies can contribute to a better understanding of their underlying effects. However, landrace designations, ambiguous common names, and gaps in sampling information complicate the identification of matching ex situ and in situ seed lots. Here we report a 50-year longitudinal comparison of the genetic diversity of a set of 13 accessions from the state of Morelos, Mexico, conserved ex situ since 1967 and retrieved in situ from the same donor families in 2017. We interviewed farmer families who donated in situ landraces to understand their germplasm selection criteria. Samples were genotyped by sequencing, producing 74,739 SNPs. Comparing the two sample groups, we show that ex situ and in situ genome-wide diversity was similar. In situ samples had 3.1% fewer SNPs and lower pairwise genetic distances (Fst 0.008–0.113) than ex situ samples (Fst 0.031–0.128), but displayed the same heterozygosity. Despite genome-wide similarities across samples, we could identify several loci under selection when comparing in situ and ex situ seed lots, suggesting ongoing evolution in farmer fields. Eight loci in chromosomes 3, 5, 6, and 10 showed evidence of selection in situ that could be related with farmers’ selection criteria surveyed with focus groups and interviews at the sampling site in 2017, including wider kernels and larger ear size. Our results have implications for ex situ collection resampling strategies and the in situ conservation of threatened landraces.