The potential of molecular cytogenetics was recognized in the plant breeding sector, often with a focus on hybridization, introgressions and chromosomal additions. Now, with the advances in long-read sequencing and with the new genome-editing techniques in place, the plant cytogenetics field is repositioning itself at the interface between microscopy and genomics. Fluorescence in situ hybridization (FISH) methods are more relevant than ever, for example to better resolve the inaccessible, repetitive genome fraction, the genomes’ three-dimensional organization, but also to identify chromosomes, assess haplotypes, predict recombination, and determine genomic variation among individuals. As most FISH experiments require spreads of mitotic chromosomes, typically, dividing plant cells are harvested. In plants, mitotic cells occur in the meristems, out of which the root apical meristems are best accessible. Hence, the wide majority of plant cytogenetics labs uses root tip tissues to prepare mitotic chromosomes, with primary roots being especially serviceable.