Extracellular vesicles (EVs) are heterogeneous populations of molecules intensively studied for their potential role as diagnostic biomarkers. EVs participate in intercellular communication through transfer of molecular cargos including microRNAs (miRNAs). Despite their roles in cell-to-cell communication, understanding of the different populations of EVs and their secretion mechanisms remains far from comprehensive. While CD9, CD63, and CD81 are members of the tetraspanin family commonly used as EV markers, it became clear that tetraspanins were not equally expressed in all EVs. However, reliable isolation of EVs represents a considerable challenge. To address this knowledge gap, EV RNA extracted from six different cell types (DiFi, placental explant, BeWo, Kuramochi, iPSC-cardiomyocytes, and serum) were obtained by immunomagnetic separation, either alone or in combination with density gradient chromatography (DGUC) and ultracentrifugation combined with density gradient (DGUC+SEC). Expression levels and distributions of tetraspanin and placental specific marker placental alkaline phosphatase (PLAP) on EVs were analyzed by small RNA sequencing. This study showed that immunomagnetic separation seemed to preferentially isolate specific subtypes of miRNA-rich EVs with high efficiencies. DGUC was able to separate EV with decreased yield and second-step purification by SEC did not improve EV separation. Moreover, our data demonstrated that tetraspanin distribution is unique for different sources of EVs. DiFi and placental explant supernatant (PES) exhibited different patterns of miRNA expression in CD9/CD63/CD81 derived EVs. All three tetraspanins were clustered together in DiFi EVs, while CD63 displayed significantly higher expression than CD9 and CD81 in PES EVs. We also discussed challenges associated with insufficient source materials, bead binding capacity and specificity. Overall, this work provides evidence for understanding of EV heterogeneity and opens up for further evaluation of EVs as promising biomarker and therapeutic targets for various diseases.