Mollicutes are the smallest organisms capable of autonomous self-replication, and their phenotype differs from that of other bacteria in their lack of a cell wall. Mollicutes are wide spread in nature and pathogenically infect humans, other mammals, reptiles, arthropods and plants. Normally demonstrate a strict specificity to a host, as well as tissue specificity, which result from an obligate parasitic lifestyle. However, plant pathogens, including phytoplasmas, spiroplasmas and acholeplasmas, have the capability to infect a variety of plants and use insect-host vectors for transmission. The objective was detection and characterization of spiroplasmas by PCR, sequencing 16S rRNA, and atomic force microscopy, in isolates of Apis mellifera in Puebla-Mexico. Samples from 100 honey bee were collected and examined by PCR, sequencing 16S rRNA and atomic force microscopy. A 271 bp product was produced from the F28/R5 primer set in 38%. Alignment of the sequences showed a 95% similarity with Spiroplasma melliferum. Atomic force microscopy study shows the presence of spherical structures formed chains with sizes within the ranges reported for spiroplasmas.