Considering the contagious nature, high mortality, quarantine and to initiate supportive treatment and patient management, accurate diagnosis of Canine Distemper (CD) infection at an early stage is essential. From 45 dogs suspected of CD, a total of 135 samples comprising of blood, nasal (NS) and conjunctival swabs (CS), were processed for lymphocyte count and RT-PCR. CD was detected in ten dogs (22.22%)by RT-PCR. The sensitivity of CS was found highly significant in distemper positive dogs (p < 0.005) followed by NS and plasma, whereas, it was insignificant (p > 0.05) when compared from distemper suspected dogs. All the distemper positive dogs were found lymphopenic. The difference in lymphocyte count in distemper positive and negative dogs was insignificant (p = 0.203). It was, however, significant (p = 0.0056) in distemper positive dogs when repeated thrice at an interval of ten days. In conclusion, lymphopenia is the suggestive of clinical infection of CD, however, it is not necessary that all lymphopenic dogs are distemper positive. In this context, RT-PCR is a test of choice with CS followed by NS swabs and plasma.