Heart Rate Variability (HRV) is extensively used to investigate general Autonomic Nervous System (ANS) function and is affected by many factors including age, gender, pathology such as diabetes and genetic polymorphisms. One of these genetic polymorphisms is the Angiotensin Converting Enzyme (ACE) polymorphism corresponding to insertion (I) or deletion (D) of a 287-base pair sequence of DNA in intron 16 of the ACE gene (rs4340). Some studies have addressed the relationship between HRV and D/D, I/D and I/I ACE polymorphism while others combined I/D and I/I ACE groups. In this study HRV is determined for diabetic and control individuals with different ACE polymorphism considering either separate or combined I/D and I/I genotypes. Linear time domain parameters, entropy, low frequency and total power of HRV were found to be significantly different between diabetic and control individuals with combined I/I and I/D ACE polymorphism, while only entropy was different for diabetic and control subjects with D/D ACE genotype. Separate analysis of I/I and I/D genotypes was preferred for a thorough investigation of HRV and ACE polymorphism, as the combined analysis masked some differences in HRV parameters such as Poincaré plot between ACE polymorphisms and diabetes status. Furthermore, a separate analysis demonstrated that most of the significant differences for HRV were between the diabetic group with I/I genotype and I/D and D/D groups.