Testicular fine needle aspiration (TFNA) has proven to be a simple and minimally invasive procedure, which allows assessments of cytological parameters of seminiferous epithelium/tubules more accurately in a short time. Though this technique does not cause negative effects on sperm quality or any damage to testicular tissue, its use is very limited in male animal infertility diagnostics. Report on the use of this technique in South American Camelids (SAC) is very limited. Therefore, the aim of this study was to evaluate the efficacy of TFNA for identification of different testicular cells and cell indices, and their correlation with that of impression cytology. A total of 98 slides were prepared from testes of six adult alpaca males, collected immediately after slaughter. Aspiration samples were performed by inserting a fine butterfly needle (21. G) connected to a 50. ml syringe into a testicle and multiple plane aspirations were carried out to obtain the materials destined to the smear. Three different imprints on slides were taken from each testicle. All slides were air-dried, stained with modified May-Grünwald-Giemsa (MGG) stain and then examined under light microscope with 1000× magnifications. Spermatogenic cells such as, spermatogonia (Sg), primary spermatocytes, secondary spermatocytes, early spermatids (ab), late spermatids (cd) and spermatozoa, and Sertoli cells were counted. The spermatozoa percentage was expressed as spermatic index (SI) and the number of Sertoli cells, counted apart, was expressed as sertoli cell index (SEI). There was not any significant difference between the spermatogenic cell parameters obtained from the two types of slides, but SEI were significantly different in two types of smears. The results of the study provide support for the use of TFNA as a useful minimally invasive modality to identify different spermatogenetic cell classes in alpaca. Moreover, the possibility to standardize this method might provide a greater impulse to the clinical diagnostics of SAC male infertility. © 2011 Elsevier B.V.