Plasmodesmata are small channels that connect plant cells. While recent technological advances have facilitated the analysis of the ultrastructure of these channels, there are limitations to efficiently addressing their presence over an entire cellular interface. Here, we highlight the value of serial block electron microscopy for this purpose. We developed a computational pipeline to study plasmodesmata distributions and we detect presence/absence of plasmodesmata clusters, pit fields, at the phloem unloading interfaces of Arabidopsis thaliana roots. Pit fields can be visualised and quantified. As the wall environment of plasmodesmata is highly specialised we also designed a tool to extract the thickness of the extracellular matrix at and outside plasmodesmata positions. We show and quantify clear wall thinning around plasmodesmata with differences between genotypes, namely in the recently published plm-2 sphingolipid mutant. Our tools open new avenues for quantitative approaches in the analysis of symplastic trafficking. We developed computational tools for serial block electron microscopy datasets to extract information on the spatial distribution of plasmodesmata over an entire cellular interface and on the wall environment the plasmodesmata are in.