Buffalo is a major dairy animal in many countries of the world, including Pakistan. However, the productive and reproductive performance of this species is affected by many physiological problems such as anestrous. Therefore, this study was conducted to compare serum total cholesterol, thyroid hormone concentrations and haematological variables in cyclic and acyclic Nili-Ravi buffaloes. For this purpose, 60 adult Nili-Ravi buffaloes were divided into two equal groups i.e. cyclic and acyclic, depending upon the presence or absence of active corpus luteum on the ovaries. Blood samples with and without anticoagulant were collected from each animal. The blood samples without anticoagulant were used for separation of serum, which was utilized for estimation of serum total cholesterol, T3 and T4, using appropriate kit method. The samples containing anticoagulant were used for determination of haematological variables viz. RBC count, Hb, PCV, ESR, MCV, MCH, MCHC, TLC, DLC and platelets count. Results showed that serum total cholesterol (142.85±7.43 V 88.84±5.53 mg/dl) was higher in cyclic than acyclic buffaloes (P≤0.05), while levels of T3 and T4 did not differ between the two groups. Among haematological variables, RBC count (6.29±0.97 V 4.87±1.62×106/μl), Hb concentration (11.54±1.61 V 9.89±1.14 g/dl), PCV (40.28±6.06 V 36.80±3.0%), MCV (62.1±3.55 V 56.81±5.35 fl), MCH (21.58±5.47 V 15.99±1.84 pg) and MCHC (29.32±2.52 V 26.95±2.03 g/dl) were higher in cyclic than acyclic buffaloes (P≤0.05), while reverse was true for TLC and platelet counts. However, ESR, lymphocytes, monocytes, eosinophils and neutrophil percentages did not differ between two groups. In conclusion, low level of serum cholesterol might have been among causes of anestrous in these buffaloes, as cholesterol is the precursor of sex hormones like progesterone and estradiol, and its level was lower in acyclic than cyclic buffaloes. However, thyroid hormones do not seem to play any significant role in the occurrence of this problem.