Accurate and rapid identification of bacterial pathogens is critical for diagnosis and effective patient care. Phenotypic identification methods are subjective, time consuming and many times fail identification of certain bacteria and especially Gram-positive rods. Overseeing the need of laboratory to have reliable alternative method of identification we designed and validated in-house 16S rRNA gene sequencing and investigated it`s usefulness in identification of Gram-positive rods. This book contains detailed information on DNA sequencing steps and experience of its usage on Gram- positive rod identification. This study shows that 16S rRNA gene sequencing, which can be done in less than 10 hours for $62.53 per isolate, is more accurate, faster and, yet, cost effective comparing to conventional identification methods, especially when used for identification of Gram-positive rods with ambiguous biochemical profiles or slow growers.