Summary Two major types of Frankia strains are usually recognized, based on the ability to sporulate in‐planta: spore‐positive (Sp+) and spore‐negative (Sp−). We carried out a study of Sp+ and Sp− Frankia strains based on nodules collected on Alnus glutinosa, Alnus incana and Alnus viridis. The nodules were phenotyped using improved histology methods, and endophytic Frankia strain genotype was determined using a multilocus sequence analysis approach. An additional sampling was done to assess the relation between Sp+ phenotype frequency and genetic diversity of Frankia strains at the alder stand scale. Our results revealed that (i) Sp+ and Sp− Alnus‐infective Frankia strains are genetically different even when sampled from the same alder stand and the same host–plant species; (ii) there are at least two distinct phylogenetic lineages of Sp+ Frankia that cluster according to the host–plant species and without regard of geographic distance and (iii) genetic diversity of Sp+ strains is very low at the alder stand scale compared with Sp− strains. Difference in evolutionary history and genetic diversity between Sp+ and Sp− Frankia allows us to discuss the possible ecological role of in‐planta sporulation.